Integrin-linked kinase and its uses

ABSTRACT

Methods for isolating ILK genes are provided. The ILK nucleic acid compositions find use in identifying homologous or related proteins and the DNA sequences encoding such proteins; in producing compositions that modulate the expression or function of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as identification of cell type based on expression, and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/390,425, filed Sep. 3, 1999 now U.S. Pat. No. 6,338,958,which is a continuation of U.S. patent application Ser. No. 09/035,706,filed Mar. 5, 1998, now issued as U.S. Pat. No. 6,001,622, which is acontinuation-in-part of U.S. patent application. Ser. No. 08/955,841filed Oct. 21, 1997, now issued as U.S. Pat. No. 6,013,782, which is acontinuation-in-part of U.S. patent application Ser. No. 08/752,345,filed Nov. 19, 1996, now abandoned, which claims priority to provisionalpatent application No. 60/009,074, filed Dec. 21, 1995.

INTRODUCTION

1. Background

Proteins of the extracellular matrix (ECM) act to influence fundamentalcell and tissue behaviors. ECM regulates cell structure, growth,survival, differentiation, motility and, at the organismal level, properdevelopment. ECM proteins interact with cells via a class of cellmembrane spanning receptors called integrins. ECM acts as a biologicalsignal, where the integrin receptor is a specific transducer across thecell's plasma membrane of this signal. Integrins are also important inproliferative disorders, mediating such processes as wound healing andinflamnmation, angiogenesis, as well as tumor migration and invasion.

A major biochemical response to ECM integrin interactions is elevationof an enzymatic activity known as protein Phosphorylation.Phosphorylation is important in signal transduction mediated byreceptors for extracellular biological signals such as growth factors orhormones. For example, many cancer causing genes (oncogenes) are proteinkinases, enzymes that catalyze protein phosphorylation reactions, or arespecifically regulated by phosphorylation. In addition, a kinase canhave its activity regulated by one or more distinct protein kinases,resulting in specific signaling cascades.

Research on signal transduction over the years has clearly establishedthe importance of direct, protein-protein interactions in the cytoplasmas a major mechanism underlying the specification of signaling pathways.These interactions can, in part, be those between a receptor and acytoplasmic protein kinase, or between a protein kinase and itssubstrate molecule(s).

A number of known protein kinases, such as mitogen-activated kinase(MAPK), focal adhesion kinase (FAK), and protein kinase C (PKC), havetheir kinase activity stimulated by integrin-ECM interaction. Forexample, see Maguire et al. (1995) J Exp Med 182:2079-2090; Richardsonand Parsons (1995) Bioessays 17:229-236; Morino et al. (1995) J. Biol.Chem. 270:269-273; and Nojima et al. (1995) J Biol Chem 270:15398-15402.However, no cellular protein kinase has been identified to date that hasbeen demonstrated to bind to an integrin molecule under physiologicalconditions. As such is the case, the direct molecular connection betweenintegrins and the ECM-induced phosphorylation of cellular proteins isunclear. As such is the case, if the direct molecular connection betweenintegrins and the ECM-induced phosphorylation of cellular proteins weredetermined, products which modulated that connection would be usefultherapeutics. These products could be used to modulate cell growth, celladhesion, cell migration and cell invasion.

It is known that kinases can form complex signaling cascades, where theactivation of one kinase causes it to activate or de-activate anotherkinase, and so forth through several iterations. One advantage to thistype of pathway is that a single “second messenger” can affect a numberof different processes, depending on the specific kinase expressionpattern in a cell. A particularly interesting second messenger in thisrespect is phosphatidylinositol 3,4,5 triphosphate [Ptdlns(3,4,5)P₃].[Ptdlns(3,4,5)P₃] acts on pathways that control cell proliferation, cellsurvival and metabolic changes—often through protein kinases. This lipidcan be produced by PI3 kinases, a family of related proteins (Vanhaesebroeck et al. (1997) TIBS 22:267; Toker and Cantley (1997) Nature387:673676). One downstream effector is protein kinase B (PKB/AKT)(Downward (1998) Science 279:673-674). PKB contains a pleckstrinhomology (PH) domain, to which the [Ptdlns(3,4,5)P₃] signaling moleculebinds. In addition, PKB itself is phosphorylated when [Ptdlns(3,4,5)P₃]is present, by two different protein kinases, one of which has beencloned (Stephens et al. (1998) Science 279:710-714; Alessi et al. (1997)Curr. Biol. 7:776). The molecular identity of the other kinase has notpreviously been established. The determination of this kinase, as wellas its substrates and modulators, is of great interest for providing apoint of intervention in this pathway.

If it were determined that a specific kinase regulates integrinfunction, products that regulate the activity of that kinase could beused for the treatment of cancer, leukemia, solid tumors, chronicinflammatory disease, restenosis, diabetes, neurological disorders,arthritis and osteoporosis, among other indications.

2. Relevant Literature

A review of integrin mediated signal transduction in oncogenesis may befound in Dedhar (1995) Cancer Metastasis Rev 14:165-172. Hannigan et al.(1995) 86th Annual Meeting of the American Institute for CancerResearch, provide a brief abstract directed to the cloning of a novelprotein kinase associated with beta integrin cytoplasmic tails. Hanniganet al. (1995) Molecular Biology of the Cell suppl. 6, p. 2244, is anabstract directed to the effect of overexpression of a novel integrinlinked kinase (ILK) in induction of a transformed phenotype and cyclinD1 expression. Rosales et al. (1995) Biochim Biophys Acta 1242:77-98reviews signal transduction by cell adhesion receptors. Signaling bycell adhesion receptors may, involve aspects that impinge on previouslyknown signaling pathways including the RTK/Ras pathway and serpentinereceptor/G protein pathways. A possible signaling role for the Syktyrosine kinase is described in Lin et al. (1995) J Biol Chem270:16189-16197.

Miyamoto et al. (1995) Science 267:883-885 compare the roles of receptoroccupancy and aggregation on integrin receptor mediation of celladhesion, signal transduction, and cytoskeletal organization. An ESTsequence is provided by EMBL sequence DNA library accession no. pH70160, the Wash. U.—Merck EST project.

The sequences of a number of kinases are known in the art, includinghuman protein kinase B (Coffer and Woodgett(1991) Eur. J. Biochem.201:475-481). PI3 kinases have been characterized, includingphosphatidylinositol 3-kinase gamma polypeptide, (OMIM 601232);phosphatidylinositol 3-kinase alpha polypeptide (OMIM 171834);phosphatidylinositol 3-kinase regulatory subunit (OMIM 171833); mousePI3 kinase (Genbank M60651); rat PI3 kinase (Genbank D78486, D64045).Glycogen synthase kinase 3 sequences can be accessed at Genbank; thehuman cDNA sequence has the accession number L40027.

SUMMARY OF THE INVENTION

Isolated nucleotide compositions and sequences are provided for integrininked kinase (ILK) genes. The ILK nucleic acid compositions find use inidentifying homologous or related genes; for production of the encodedkinase; in producing compositions that modulate the expression orfunction of its encoded protein; for gene therapy; mapping functionalregions of the protein; and in studying associatedphysiological,pathways. In addition, modulation of the gene activity invivo is used for prophylactic and therapeutic purposes, such astreatment of cancer, identification of cell type based on expression,and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A(i,ii)-FIG. 1D Yeast two-hybrid cloning, characterization, andexpression of ILK. a. Homology with protein kinase subdomains I to XI.b, Amino acid residues comprising ankyrin repeats. c, BIT-9 used toprobe RNA from human tissues. d, Analysis of whole cell lysates ofmouse, rat and human cell lines.

FIG. 2A-FIG. 2C In vitro and immune-complex kinase assays. a, In vitrokinase reactions. b, Immune complexes. c, ³²P-labeled products isolatedand analyzed for phosphoamino acid content.

FIG. 3A-FIG. 3D Antibodies to GST-ILK²³² recognize p59_(ILK) in integrinco-immunoprecipitations. a, Unfractionated polyclonal anti-ILK seraspecifically recognize a ³⁵ S-methionine, metabolically-labeled cellularprotein. b, Affinity-purified antibody was adsorbed with GST-ILKagarose-GST. c, Polyclonal anti-integrin antibodies used to precipitatesurface-biotinylated integrins from PC3 cells. d, Anti-1 monoclonalantibodies were used in co-precipitation analyses of lysates of PC3.

FIG. 4A-FIG. 4E Modulation of ILK kinase activity by ECM components. a,ILK phosphorylation of MBP was assayed. b, Expression levels of p59ILK.c, Representative p59_(ILK) overexpressing clone ILK13-A4a on the ECMsubstrates. d, Adhesion of the ILK overexpressing clones to LN, FN andVN was quantified. e, ILK13, p59_(ILK) overexpressing clones wereassayed for colony growth.

FIG. 5A-FIG. 5D Expression of ILK in human breast carcinomas. a, Normalregion of breast tissue. b, Ductal carcinoma in situ. c,d, lnvasivecarcinoma.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Nucleic acid compositions encoding integrin linked kinase (ILK) areprovided. They are used in identifying homologous or related genes; inproducing compositions that modulate the expression or function of itsencoded protein; for gene therapy; mapping functional regions of theprotein; and in studying associated physiological pathways. The ILK geneproduct (herein. p59ILK) is a serine threonine kinase having twofunctional domains, identified by comparison of the ILK sequence againstthose found in current protein databases. These are the catalyticdomain, responsible for phosphotransferase activity (kinase domain), anda non-overlapping domain in the amino terminus, comprised of fourcontiguous ankyrin-like repeats.

Modulation of ILK gene activity in vivo is used for prophylactic andtherapeutic purposes, such as treatment of cancer, investigation ofintegrin signaling pathway function, identification of cell type basedon expression, and the like. The protein is useful as an immunogen forproducing specific antibodies, in screening for biologically activeagents that act in the integrin signaling pathway and for therapeuticand prophylactic purposes.

The present invention demonstrates a physical linkage between integrinand ILK. Dysregulated expression of ILK protein modulates the functionof integrins, thus providing a biological link between ILK and integrin.Dysregulated expression of ILK modulates cell growth, cell adhesion,cell migration and cell invasion. Hence, products that modulate theexpression and/or activity of ILK have a therapeutic effect in thetreatment of cancer, leukemia, solid tumors, chronic or acuteinflammatory disease, restenosis, diabetes, neurological disorders,arthritis and osteoporosis, among other indications.

Characterization of ILK

The human gene sequence of ILK is provided as SEQ ID NO:1, the encodedpolypeptide product as SEQ ID NO:2. The ILK protein is encoded by a 1.8kilobase pair messenger RNA (1.8 kb mRNA). The sequence of this mRNA wasused to deduce the primary amino acid sequence of the protein, which hasa predicted molecular weight of 50 kiloDaltons (kDa). The recombinantprotein migrates on analytical polyacrylamide electrophoresis gels withan apparent molecular weight of 59 kDa, in rough agreement with thepredicted size. p59ILK is a serine threonine kinase having twofunctional domains, identified by comparison of the ILK sequence againstthose found in current protein databases. These are the -catalyticdomain, responsible for phosphotransferase activity (kinase domain), anda non-overlapping domain in the amino terminus, comprised of fourcontiguous ankyrin-like repeats.

The function of ankyrin repeats in ILK is to mediate protein-proteininteractions. The ILK ankyrin repeat domain is not required for thebinding of p59_(ILK) to integrin, and it is predicted to mediate theinteraction of p59_(ILK) with other cellular protein(s). Thus, p59_(ILK)bridges integrin in the plasma membrane with intracellular proteinsactive regulating the cell's response to ECM signals. These proteins arelikely to be located in the cytoplasm, or as part of the cell'sstructural framework (cytoskeleton).

ILK has novel structural and functional features. The moleculararchitecture is unusual, in that a protein kinase and an ankyrin repeatdomain are contained within the same protein. The kinase domain has ahigh degree of similarity to other kinase sequences in existingdatabases, and can be divided into typical subdomains (I through XI)based on this conserved structure. However one amino acid in subdomainVIb of all other protein kinase domains is not present in ILK. Despitethis unique structural feature, ILK clearly acts as a protein kinase,and thus represents a prototype member of a new subfamily of proteinkinase molecules.

ILK regulates integrin extracellular activity (ECM interactions) frominside the cell via its direct interaction with the integrin subunit(colloquially known as inside-out signaling). Interfering with ILKactivity allows the specific targeting of integrin function, whileleaving other essential signaling pathways intact. Moreover, increasingthe levels of cellular ILK activity short circuits the normalrequirement for adhesion to ECM (i.e. integrin function) in regulatingcell growth. Thus, inhibiting ILK activity inhibitsanchorage-independent (i.e. cancerous) cell growth.

The amino acid sequence of ILK contains a sequence motif found inpleckstrin homology (PH) domains (Klarulund et al. (1997) Science275:1927-1930). This motif has been shown to be involved in the bindingof phosphatidylinositol phosphates (Lemmon et al. (1996) Cell85:621-624). Amino acids critical to the binding of such lipids to thePH domain are completely conserved in ILK. The phosphatidylinositol3,4,5, triphosphate binding sites are the lysines at positions 162 and209 (SEQ ID NO:2). The PH motifs are comprised of residues 158-165 and208-212 (SEQ ID NO:2). There is a high degree of sequence identitywithin this motif between ILK and other PH-domain containing proteinssuch as cytohesin-1 (a β2 integrin cytoplasmic domain interactingprotein) and GRP-1. It was determined that ILK activity is influenced bythe presence of phosphatidylinositol3,4,5, triphosphate, and interactswith other kinase proteins in this pathway.

ILK activity can be stimulated by phosphatidylinositol 3,4,5trisphosphate in vitro. Both insulin and fibronectin can rapidlystimulate ILK activity in a phosphoinositide-3OH kinase(PI(3)K)-dependent manner. In addition, constitutively active PI(3)Kactivates ILK. The activated ILK can then inhibit the activity ofglycogen synthase kinase-3 (GSK-3), contributing to ILK induced nucleartranslocation of β-catenin. ILK can also phosphorylate protein kinase B(PKB/AKT) on serine 473, resulting in its activation, demonstrating thatILK is involved in agonist stimulated PI(3)K-dependent PKB/AKTactivation.

In untransformed intestinal epithelial cells, the kinase activity of ILKis inhibited upon cell-extracellular matrix interactions, andoverexpression of constitutively active ILK results inanchorage-independent growth and tumorigenicity in nude mice. Aconsequence of elevation of ILK levels is a disruption of cell-cellinteractions and manifestation of fibroblastic cell morphology andphenotypic properties, which include formation of a fibronectin matrixand invasion of collagen gels.

Overexpression of ILK results in a downregulation of E-cadherinexpression, formation of a complex between β-catenin and the HMGtranscription factor, LEF-1, translocation of β-catenin to the nucleus,and transcriptional activation by this LEF-1/β-catenin complex. LEF-1protein expression is rapidly modulated by cell detachment from theextracellular- matrix, and LEF-1 protein levels are constitutivelyupregulated upon ILK overexpression. These effects are specific for ILK.

Overexpression of ILK stimulates fibronectin matrix assembly inepithelial cells. The integrin-linked kinase activity is involved intransducing signals leading to the up-regulation of fibronectin matrixassembly, as overexpression of a kinase-inactive ILK mutant fails toenhance the matrix assembly. The increase in fibronectin matrix assemblyis accompanied by a substantial reduction in cellular E-cadherin. Theincreased fibronectin matrix assembly is associated with an increasedpotential for tumor growth in vitro and in vivo.

The ILK chromosomal locus is mapped to region 11p15. A subset of breastcarcinomas displays LOH for markers in chromosomal region 11p15.5. Thisregion has also been implicated in an inherited form of cardiacarrhythmia, the long QT syndrome. A high level of expression of ILK MRNAindicates an integrin-independent function for ILK in cardiac tissue.

The genomic sequence of human integrin-linked kinase is provided as SEQID NO:12. There are 13 exons in the ILK gene, having the positions: SEQID NO:12: (1-41); (401-581); (4260-4425); (4608-4703); (4908-5004);(5098-5181); (5285-5370); (5513-5622); (5726-5853); (5938-6059);(6150-6249); (6362-6492); and (6676-7080). The genomic sequences finduse for producing the protein in eukaryotic expression systems; indesigning primers e.g. for microsatellite analysis, PCR amplification,etc.; for providing regions of homology for integration into the hostgenome; as a hybridization probe; and the like.

Identification of ILK Sequences

Homologs of ILK are identified by any of a number of methods. A fragmentof the provided cDNA may be used as a hybridization probe against a cDNAlibrary from the target organism of interest, where low stringencyconditions are used. The probe may be a large fragment, or one or moreshort degenerate primers. Nucleic acids having sequence similarity aredetected by hybridization under low stringency conditions, for example,at 50° C. and 10×SSC (0.9 M saline/0.09 M sodium citrate) and remainbound when subjected to washing at 55° C. in 1×SSC. Sequence identitymay be determined by hybridization under stringent conditions, forexample, at 50° C. or higher and 0.1×SSC (9 mM saline/0.9 mM sodiumcitrate). Nucleic acids that are substantially identical to the providedILK sequences, e.g. allelic variants, genetically altered versions ofthe gene, etc., bind to the provided ILK sequences under stringenthybridization conditions. By using probes, particularly labeled probesof DNA sequences, one can isolate homologous or related genes. Thesource of homologous genes may be any species, e.g. primate species,particularly human; rodents, such as rats and mice, canines, felines,bovines, ovines, equines, yeast, nematodes, etc.

Between mammalian species, e.g. human and mouse, homologs havesubstantial sequence similarity, i.e. at least 75% sequence identitybetween nucleotide sequences. Sequence similarity is calculated based ona reference sequence, which may be a subset of a larger sequence, suchas a conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. (1990) J Mol Biol215:403-10. The sequences provided herein are essential for recognizingILK related and homologous proteins in database searches.

ILK Nucleic Acid Compositions.

Nucleic acids encoding ILK may be CDNA or genomic DNA or a fragmentthereof. The term ILK gene shall be intended to mean the open readingframe, encoding specific ILK polypeptides, introns, as well as adjacent5 and 3 non-coding nucleotide sequences involved in the regulation ofexpression, up to about 20 kb beyond the coding region, but possiblyfurther in either direction. The gene may be introduced into anappropriate vector for extrachromosomal maintenance or for integrationinto a host genome. The term cDNA as used herein is intended to includeall nucleic acids that share the arrangement of sequence elements foundin native mature mRNA species, where sequence elements are exons and 3and 5 non-coding regions. Normally mRNA species have contiguous exons,with the intervening introns, when present, removed by nuclear RNAsplicing, to create a continuous open reading frame encoding a ILKprotein.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include the 3 and 5 untranslatedregions found in the mature mRNA. It may further include specifictranscriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5 or 3 end of the transcribed region.The genomic DNA may be isolated as a fragment of 100 kbp or smaller; andsubstantially free of flanking chromosomal sequence. The genomic DNAflanking the coding region, either 3′ or 5′, or internal regulatorysequences as sometimes found in introns, contains sequences required forproper tissue and stage specific expression. Sequences of interest alsoinclude sequences that ross intron/exon borders, as set forth in SEQ IDNO:12.

The sequence of the 5′ flanking region may be utilized for promoterelements, including enhancer binding sites, that provide fordevelopmental regulation in tissues where ILK is expressed. The tissuespecific expression is useful for determining the pattern of expression,and for providing promoters that mimic the native pattern of expression.Naturally occurring polymorphisms in the promoter region are useful fordetermining natural variations in expression, particularly those thatmay be associated with disease.

Alternatively, mutations may be introduced into the promoter region todetermine the effect of altering expression in experimentally definedsystems. Methods for the identification of specific DNA motifs involvedin the binding of transcriptional factors are known in the art, e.g.sequence similarity to known binding motifs, gel retardation studies,etc. For examples, see Blackwell et al. (1995) Mol Med 1:194-205;Mortlock et al. (1996) Genome Res. 6:327-33; and Joulin and Richard-Foy(1995) Eur J. Biochem 232:620-626.

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of ILKexpression, especially in different tissues or stages of development,and to identify cis acting sequences and trans acting factors thatregulate or mediate ILK expression. Such transcription or translationalcontrol regions may be operably linked to a ILK gene in order to promoteexpression of wild type or altered ILK or other proteins of interest incultured cells, or in embryonic, fetal or adult tissues, and for genetherapy.

The nucleic acid compositions of the subject invention may encode all ora part of the subject polypeptides. Double or single stranded fragmentsmay be obtained of the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least 15 nt, usually at least 18 nt or 25 nt,and may be at least about 50 nt. Such small DNA fragments are useful asprimers for PCR, hybridization screening probes, etc. Larger DNAfragments, i.e. greater than 100 nt are useful for production of theencoded polypeptide. Regions of the provided sequence that are ofinterest as fragments include the 5′ end of the gene, i.e. a portion ofthe sequence set forth in SEQ ID NO:1, nucleotides 1 to 1100.

For use in amplification reactions, such as PCR, a pair of primers willbe used. The exact composition of the primer sequences is not criticalto the invention, but for most applications the primers will hybridizeto the subject sequence under stringent conditions, as known in the art.It is preferable to choose a pair of primers that will generate anamplification product of at least about 50 nt, preferably at least about100 nt. Algorithms for the selection of primer sequences are generallyknown, and are available in commercial software packages. Amplificationprimers hybridize to complementary strands of DNA, and will primetowards each other.

The ILK genes are isolated and obtained in substantial purity, generallyas other than an intact chromosome. Usually, the DNA will be obtainedsubstantially free of other nucleic acid sequences that do not include aILK sequence or fragment thereof generally being at least about 50%,usually at least about 90% pure and are typically recombinant, i.e.flanked by one or more nucleotides with which it is not normallyassociated on a naturally occurring chromosome.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature and does not require elaborationhere. DNA or mRNA is isolated from a cell sample. The mRNA may beamplified by RT-PCR, using reverse transcriptase to form a complementaryDNA strand, followed by polymerase chain reaction amplification usingprimers specific for the subject DNA sequences. Alternatively, the mRNAsample is separated by gel electrophoresis, transferred to a suitablesupport, e.g. nitrocellulose, nylon, etc., and then probed with afragment of the subject DNA as a probe. Other techniques, such asoligonucleotide ligation assays, in situ hybridizations, andhybridization to DNA probes arrayed on a solid chip may also find use.Detection of mRNA hybridizing to the subject sequence is indicative ofILK gene expression in the sample.

The sequence of a ILK gene, including flanking promoter regions andcoding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, etc. The DNA sequence or protein product of such a mutationwill usually be substantially similar to the sequences provided herein,i.e. will differ by at least one nucleotide or amino acid, respectively,and may differ by at least two but not more than about ten nucleotidesor amino acids. The sequence changes may be substitutions, insertions ordeletions. Deletions may further include larger changes, such asdeletions of a domain or exon. Other modifications of interest includeepitope tagging, e.g. with the FLAG system, HA, etc. For studies ofsubcellular localization, fusion proteins with green fluorescentproteins (GFP) may be used.

Techniques for in vitro mutagenesis of cloned genes are known. Examplesof protocols for site specific mutagenesis may be found in Gustin et al.(1993) Biotechniques 14:22; Barany (1985) Gene 37:111-23; Colicelli etal. (1985) Mol Gen Genet 199:537; and Prentki et al. (1984) Gene29:303-13. Methods for site specific mutagenesis can be found inSambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp. 15.3-15.108; Weiner et al., Gene 126:35-41 (1993); Sayers et al.Biotechniques 13:592-6 (1992); Jones and Winistorfer, Biotechniques12:528-30 (1992); Barton et al., Nucleic Acids Res 18:7349-55 (1990);Marotti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu, Anal Biochem177:1204 (1989). Such mutated genes may be used to studystructure-function relationships of ILK, or to alter properties of theprotein that affect its function or regulation.

ILK Polypeptides

The subject gene may be employed for producing all or portions of ILKpolypeptides. For expression, an expression cassette may be employed.The expression vector will provide a transcriptional and translationalinitiation region, which may be inducible or constitutive, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native toan ILK gene, or may be derived from exogenous sources.

The peptide may be expressed in prokaryotes or eukaryotes in accordancewith conventional ways, depending upon the purpose for expression. Forlarge scale production of the protein, a unicellular organism, such asE. coli, B. subtilis, S. cerevisiae, insect cells in combination withbaculovirus vectors, or cells of a higher organism such as vertebrates,particularly mammals, e.g. COS 7 cells, may be used as the expressionhost cells. In some situations, it is desirable to express the ILK genein eukaryotic cells, where the ILK protein will benefit from nativefolding and post-translational modifications. Small peptides can also besynthesized in the laboratory. Peptides that are subsets of the completeILK sequence may be used to identify and investigate parts of theprotein important for function, such as the GTPase binding domain, or toraise antibodies directed against these regions. Peptides may be fromabout 8 amino acids in length, usually at least about 12 amino acids inlength, or 20 amino acids in length, and up to complete domains, e.g.the ankyrin domains, or a substantially complete protein, i.e. 90 to 95%of the mature polypeptide.

With the availability of the protein or fragments thereof in largeamounts, by employing an expression host, the protein may be isolatedand purified in accordance with conventional ways. A lysate may beprepared of the expression host and the lysate purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique. The purified protein will generally beat least about 80% pure, preferably at least about 90% pure, and may beup to and including 100% pure. Pure is intended to mean free of otherproteins, as well as cellular debris.

The expressed ILK polypeptides are useful for the production ofantibodies, where short fragments provide for antibodies specific forthe particular polypeptide, and larger fragments or the entire proteinallow for the production of antibodies over the surface of thepolypeptide. Antibodies may be raised to the wild-type or variant formsof ILK. Antibodies may be raised to isolated peptides corresponding tothese domains, or to the native protein.

Antibodies are prepared in accordance with conventional ways, where theexpressed polypeptide or protein is used as an immunogen, by itself orconjugated to known immunogenic carriers, e.g. KLH, preS HBsAg, otherviral or eukaryotic proteins, or the like. Various adjuvants may beemployed, with a series of injections, as appropriate. For monoclonalantibodies, after one or more booster injections, the spleen isisolated, the lymphocytes immortalized by cell fusion, and then screenedfor high affinity antibody binding. The immortalized cells, i.e.hybridomas, producing the desired antibodies may then be expanded. Forfurther description, see Monoclonal Antibodies: A Laboratory Manual,Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold SpringHarbor, N.Y., 1988. If desired, the mRNA encoding the heavy and lightchains may be isolated and mutagenized by cloning in E. coli, and theheavy and light chains mixed to further enhance the affinity of theantibody. Alternatives to in vivo immunization as a method of raisingantibodies include binding to phage display libraries, usually inconjunction with in vitro affinity maturation.

Modulation of ILK Activity

ILK activity is upregulated by the presence of the lipid[Ptdlns(3,4,5)P₃]. The activity of ILK is manipulated by agents thataffect cellular levels of [Ptdlns(3,4,5)P₃ ], or that block the bindingof [Ptdlns(3,4,5)P₃] to ILK. This lipid binds to specific amino acidresidues in ILK. The amino acid sequence of ILK contains a sequencemotif found in pleckstrin homology (PH) domains, which are involved inthe binding of phosphatidylinositol phosphates. The [Ptdlns(3,4,5)P₃ ]binding sites are the lysines at positions 162 and 209 (SEQ ID NO:2).The PH motifs are comprised of residues 158-165 and 208-212 (SEQ IDNO:2).

Agents of interest for down-regulating ILK activity include directblocking of [Ptdlns(3,4,5)P₃] binding sites through competitive binding,steric hindrance, etc. Of particular interest are antibodies that bindto the PH domains, thereby blocking the site. Antibodies includefragments, e.g. F(Ab), F(Ab)′, and other mimetics of the binding site.Such antibodies can be raised by immunization with the protein or thespecific domain. Mimetics are identified by screening methods, asdescribed herein. Analogs of [Ptdins(3,4,5)P₃ ] that compete for bindingsites but do not result in activation of ILK are also of interest.

The activity of ILK is also down-regulated by inhibiting the activity ofPI(3.) kinase, thereby decreasing cellular levels of [Ptdins(3,4,5)P₃].Phosphatidylinositol 3-kinase (EC 2.7.1.137) is composed of 85-kD and110-kD subunits. The 85-kD subunit lacks PI3-kinase activity and acts asan adapter, coupling the 110-kD subunit (p110) to activated proteintyrosine kinases. p110 may require a complex with p85-alpha forcatalytic activity. The genetic and amino acid sequence of p110 subunitsfor human PI(3) kinase can be obtained from Genbank, accession numbersZ29090, X83368.

Agents of interest include inhibitors of PI(3) kinase, e.g. wortmannin,LY294002, etc. Physiologically effective levels of wortmannin range fromabout 10 to 1000 nM, usually from about 100 to 500 nM, and optimally atabout 200 nM. Physiologically effective levels of LY294002 range fromabout 1 to 500 μM, usually from about 25 to 100 μM, and optimally atabout 50 μM. The inhibitors are administered in vivo or in vitro at adose sufficient to provide for these concentrations in the targettissue.

Other inhibitors of PI(3) kinase include anti-sense reagents, asdescribed for ILK, which are specific for PI(3) kinase. Of particularinterest are anti-sense molecules derived from the human PI(3) kinasesequence, particularly the catalytic

110 subunit, using the publicly available sequence. Alternatively,antibodies, antibody fragments and analogs or other blocking agents areused to bind to the PI(3) kinase in order to reduce the activity.

Agents that block ILK activity provide a point of intervention in animportant signaling pathway. As described in other sections of theinstant application, numerous agents are useful in reducing ILKactivity, including agents that directly modulate ILK expression, e.g.expression vectors, anti-sense specific for ILK, ILK specific antibodiesand analogs thereof, small organic molecules that block ILK catalyticactivity, etc.; and agents that affect ILK activity through direct orindirect modulation of [Ptdlns(3,4,5)P₃] levels in a cell.

ILK phosphorylates protein kinase B (PKB/AKT) at amino acid residue 473,which is a serine. The sequence of PKB may be found in Genbank,accession number X61037. By modulating ILK activity, the phosphorylationof PKB ser473 is manipulated, either increasing or decreasing the level.The ser473 phosphorylation increased the catalytic activity of PKB.Modulating the activity of PKB affects the activity of GSK-3, which isinactivated by phosphorylation at ser9 (Genbank L40027). Theinactivation of GSK-3 may also be directly affected by ILK. Onceinactivated, GSK-3 results in the nuclear translocation of β-catenin andactivation of Lef-1/β-catenin transcriptional activity.

Formulations

The compounds of this invention can be incorporated into a variety offormulations for therapeutic administration. Particularly, agents thatmodulate ILK activity, or ILK polypeptides and analogs thereof areformulated for administration to patients for the treatment of ILKdysfunction, where the ILK activity is undesirably high or low, e.g. toreduce the level of ILK in cancer cells. More particularly, thecompounds of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants, gels, microspheres, and aerosols. As such,administration of the compounds can be achieved in various ways,including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration. The ILK maybe systemic after administration or may be localized by the use of animplant that acts to retain the active dose at the site of implantation.

The compounds of the present invention can be administered alone, incombination with each other, or they can be used in combination withother known compounds. In pharmaceutical dosage forms, the compounds maybe administered in the form of their pharmaceutically acceptable salts,or they may also be used alone or in appropriate association, as well asin combination with other pharmaceutically active compounds. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant is placed in proximity to the site of infection,so that the local concentration of active agent is increased relative tothe rest of the body.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Typical dosages for systemic administration range from 0.1 μg to 100milligrams per kg weight of subject per administration. A typical dosagemay be one tablet taken from two to six times daily, or one time-releasecapsule or tablet taken once a day and containing a proportionallyhigher content of active ingredient. The time-release effect may beobtained by capsule materials that dissolve at different pH values, bycapsules that release slowly by osmotic pressure, or by any other knownmeans of controlled release.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

The use of liposomes as a delivery vehicle is one method of interest.The liposomes fuse with the cells of the target site and deliver thecontents of the lumen intracellularly. The liposomes are maintained incontact with the cells for sufficient time for fusion, using variousmeans to maintain contact, such as isolation, binding agents, and thelike. In one aspect of the invention, liposomes are designed to beaerosolized for pulmonary administration. Liposomes may be prepared withpurified proteins or peptides that mediate fusion of membranes, such asSendai virus or influenza virus, etc. The lipids may be any usefulcombination of known liposome forming lipids, including cationic lipids,such as phosphatidylcholine. The remaining lipid will normally beneutral lipids, such as cholesterol, phosphatidyl serine, phosphatidylglycerol, and the like.

For preparing the liposomes, the procedure described by Kato et al.(1991) J. Biol. Chem. 266:3361 may be used. Briefly, the lipids andlumen composition containing the nucleic acids are combined in anappropriate aqueous medium, conveniently a saline medium where the totalsolids will be in the range of about 1-10 weight percent. After intenseagitation for short periods of time, from about 5-60 sec., the tube isplaced in a warm water bath, from about 25-40° C. and this cyclerepeated from about 5-10 times. The composition is then sonicated for aconvenient period of time, generally from about 1-10 sec. and may befurther agitated by vortexing. The volume is then expanded by addingaqueous medium, generally increasing the volume by about from 1-2 fold,followed by shaking and cooling. This method allows for theincorporation into the lumen of high molecular weight molecules.

Diagnostic Uses

DNA-based reagents derived from the sequence of ILK, e.g. PCR primers,oligonucleotide or cDNA probes, as well as antibodies against p59ILK,are used to screen patient samples, e.g. biopsy-derived tumors,inflammatory samples such as arthritic synovium, etc., for amplified ILKDNA, or increased expression of ILK mRNA or protein. DNA-based reagentsare designed for evaluation of chromosomal loci implicated in certaindiseases e.g. for use in loss-of-heterozygosity (LOH) studies, or designof primers based on ILK coding sequence.

The subject nucleic acid and/or polypeptide compositions may be used toanalyze a patient sample for the presence of polymorphisms associatedwith a disease state or genetic predisposition to a disease state.Biochemical studies may be performed to determine whether a sequencepolymorphism in an ILK coding region or control regions is associatedwith disease, particularly cancers and other growth abnormalities.Diseases of interest may also include restenosis, diabetes, neurologicaldisorders, etc. Disease associated polymorphisms may include deletion ortruncation of the gene, mutations that alter expression level, thataffect the binding activity of the protein to integrin, kinase activitydomain, etc.

Changes in the promoter or enhancer sequence that may affect expressionlevels of ILK can be compared to expression levels of the normal alleleby various methods known in the art. Methods for determining promoter orenhancer strength include quantitation of the expressed natural protein;insertion of the variant control element into a vector with a reportergene such as β-galactosidase, luciferase, chloramphenicolacetyltransferase, etc. that provides for convenient quantitation; andthe like.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence, e.g. a disease associated polymorphism.Where large amounts of DNA are available, genomic DNA is used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. Cells that express ILKmay be used as a source of mRNA, which may be assayed directly orreverse transcribed into cDNA for analysis. The nucleic acid may beamplified by conventional techniques, such as the polymerase chainreaction (PCR), to provide sufficient amounts for analysis. The use ofthe polymerase chain reaction is described in Saiki, et al. (1985)Science 239:487, and a review of techniques may be found in Sambrook, etal. Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp.14.2-14.33. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al. (1990) N.A.R. 18:2887-2890; and Delahuntyet al. (1996) Am. J. Hum. Genet. 58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin,allophycocyanin,6-carboxyfluorescein(6-FAM),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein(JOE), 6-carboxy-X-rhodamine (ROX),6-carboxy-2,4,7,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N,N-tetramethyl-6-carboxyrhodamine. (TAMRA), radioactivelabels, e.g. ³2 P, ³5 S, ³ H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g. amplified or cloned fragment, is analyzedby one of a number of methods known in the art. The nucleic acid may besequenced by dideoxy or other methods, and the sequence of basescompared to a wild-type ILK sequence. Hybridization with the variantsequence may also be used to determine its presence, by. Southern blots,dot blots, etc. The hybridization pattern of a control and variantsequence to an array of oligonucleotide probes immobilised on a solidsupport, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, mayalso be used as a means of detecting the presence of variant sequences.Single strand conformational polymorphism (SSCP) analysis, denaturinggradient gel electrophoresis(DGGE), and heteroduplex analysis in gelmatrices are used to detect conformational changes created by DNAsequence variation as alterations in electrophoretic mobility.Alternatively, where a polymorphism creates or destroys a recognitionsite for a restriction endonuclease, the sample is digested with thatendonuclease, and the products size fractionated to determine whetherthe fragment was digested. Fractionation is performed by gel orcapillary electrophoresis, particularly acrylamide or agarose gels.

Screening for mutations in ILK may be based on the functional orantigenic characteristics of the protein. Protein truncation assays areuseful in detecting deletions that may affect the biological activity ofthe protein. Various immunoassays designed to detect polymorphisms inILK proteins may be used in screening. Where many diverse geneticmutations lead to a particular disease phenotype, functional proteinassays have proven to be effective screening tools. The activity of theencoded ILK protein in kinase assays, binding of integrins, etc., may bedetermined by comparison with the wild-type protein.

Antibodies specific for a ILK may be used in staining or inimmunoassays. Samples, as used herein, include biological fluids such assemen, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluidand the like; organ or tissue culture derived fluids; and fluidsextracted from physiological tissues. Also included in the term arederivatives and fractions of such fluids. The cells may be dissociated,in the case of solid tissues, or tissue sections may be analyzed.Alternatively a lysate of the cells may be prepared.

Diagnosis may be performed by a number of methods to determine theabsence or presence or altered amounts of normal or abnormal ILK inpatient cells. For example, detection may utilize staining of cells orhistological sections, performed in accordance with conventionalmethods. Cells are permeabilized to stain cytoplasmic molecules. Theantibodies of interest are added to the cell sample, and incubated for aperiod of time sufficient to allow binding to the epitope, usually atleast about 10 minutes. The antibody may be labeled with radioisotopes,enzymes, fluorescers, chemiluminescers, or other labels for directdetection. Alternatively, a second stage antibody or reagent is used toamplify the signal. Such reagents are well known in the art. Forexample, the primary antibody may be conjugated to biotin, withhorseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to aflourescent compound, e.g. flourescein rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

Diagnostic screening may also be performed for polymorphisms that aregenetically linked to a disease predisposition, particularly through theuse of microsatellite markers or single nucleotide polymorphisms.Frequently the microsatellite polymorphism itself is not phenotypicallyexpressed, but is linked to sequences that result in a diseasepredisposition. However, in some cases the microsatellite sequenceitself may affect gene expression. Microsatellite linkage analysis maybe performed alone, or in combination with direct detection ofpolymorphisms, as described above. The use of microsatellite markers forgenotyping is well documented. For examples, see Mansfield et al. (1994)Genomics 24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031; Dib etal., supra.

Diagnostic screening may also be performed for polymorphisms that aregenetically linked to a predisposing mutation, particularly through theuse of microsatellite markers or single nucleotide polymorphisms.Frequently the microsatellite polymorphism itself is not phenotypicallyexpressed, but is linked to sequences that result in a diseasepredisposition. However, in some cases the microsatellite sequenceitself may affect gene expression. Microsatellite linkage analysis maybe performed alone, or in combination with direct detection ofpolymorphisms, as described above. The use of microsatellite markers forgenotyping is well documented. For examples, see Mansfield et al. (1994)Genomics 24:225-233; Ziegle et al. (1992).Genomics 14:1026-1031; Dib etat., supra.

Microsatellite loci that are useful in the subject methods have thegeneral formula:

U (R)_(n)U′,

where

U and U′ are non-repetitive flanking sequences that uniquely identifythe particular locus, R is a repeat motif, and n is the number ofrepeats. The repeat motif is at least 2 nucleotides in length, up to 7,usually 2-4 nucleotides in length. Repeats can be simple or complex. Theflanking sequences U and U′ uniquely identify the microsatellite locuswithin the human genome. U and U′ are at least about 18 nucleotides inlength, and may extend several hundred bases up to about 1 kb on eitherside of the repeat. Within U and U′, sequences are selected foramplification primers. The exact composition of the primer sequences arenot critical to the invention, but they must hybridize to the flankingsequences U and U′, respectively, under stringent conditions. Criteriafor selection of amplification primers are as previously discussed. Tomaximize the resolution of size differences at the locus, it ispreferable to chose a primer sequence that is close to the repeatsequence, such that the total amplification product is between 100-500nucleotides in length.

The number of repeats at a specific locus, n, is polymorphic in apopulation, thereby generating individual differences in the length ofDNA that lies between the amplification primers. The number will varyfrom at least 1 repeat to as many as about 100 repeats or more.

The primers are used to amplify the region of genomic DNA that containsthe repeats. Conveniently, a detectable label will be included in theamplification reaction, as previously described. Multiplex amplificationmay be performed in which several sets of primers are combined in thesame reaction tube. This is particularly advantageous when limitedamounts of sample DNA are available for analysis. Conveniently, each ofthe sets of primers is labeled with a different fluorochrome.

After amplification, the products are size fractionated. Fractionationmay be performed by gel electrophoresis, particularly denaturingacrylamide or agarose gels. A convenient system uses denaturingpolyacrylamide gels, in combination with an automated DNA sequencer, seeHunkapillar et al. (1991) Science 254:59-74. The automated sequencer isparticularly useful with multiplex amplification or pooled products ofseparate PCR reactions. Capillary electrophoresis may also be used forfractionation. A review of capillary electrophoresis may be found inLanders, et al. (1993) BioTechniques 14:98-111. The size of theamplification product is proportional to the number of repeats (n) thatare present at the locus specified by the primers. The size will bepolymorphic in the population, and is therefore an allelic marker forthat locus.

Modulation of Gene Expression

From a therapeutic point of view, inhibiting ILK activity has atherapeutic effect on a number of proliferative disorders, includinginflammation, restenosis, and cancer. Inhibition is achieved in a numberof ways. Antisense ILK sequences may be administered to inhibitexpression. Pseudo-substrate inhibitors, for example, a peptide thatmimics a substrate for ILK may be used to inhibit activity. Otherinhibitors are identified by screening for biological activity in anILK-based functional assay, e.g. in vitro or in vivo ILK kinaseactivity.

The ILK genes, gene fragments, or the encoded protein or proteinfragments are useful in gene therapy to treat disorders associated withILK defects. Expression vectors may be used to introduce the ILK geneinto a cell. Such vectors generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences. Transcription cassettes may be preparedcomprising a transcription initiation region, the target gene orfragment thereof, and a transcriptional termination region. Thetranscription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained in thecells, usually for a period of at least about one day, more usually fora period of at least about several days to several weeks.

The gene or ILK protein may be introduced into tissues or host cells byany number of routes, including viral infection, microinjection, orfusion of vesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al. (1992) Anal Biochem205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al. (1992)Nature 356:152-154), where gold microprojectiles are coated with the ILKor DNA, then bombarded into skin cells.

Antisense molecules can be used to down-regulate expression of ILK incells. The anti-sense reagent may be antisense oligonucleotides (ODN),.particularly synthetic ODN having chemical modifications from nativenucleic acids, or nucleic acid constructs that express such anti-sensemolecules as RNA. The antisense sequence is complementary to the mRNA ofthe targeted gene, and inhibits expression of the targeted geneproducts. Antisense molecules inhibit gene expression through variousmechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is a.synthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996) Nature Biotechnology 14:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in vitro or in an animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner. et al. (1993) supra. and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O-5′-S-phosphorothioate, 3′-S′-50-phosphorothioate,3′-CH₂-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5′-methyl-2′-deoxycytidine and 5′-bromo-2′-deoxycytidinefor deoxycytidine. 5′-propynyl-2′-deoxyuridine and5′-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995) Nucl. Acids Res 23:4434-42). Examples of oligonucleotideswith catalytic activity are described in. WO 9506764. Conjugates ofanti-sense ODN with a metal complex, e.g. terpyridylCu(II), capable ofmediating MRNA hydrolysis are described in Bashkin et al. (1995) ApplBiochem Biotechnol 54:43-56.

Genetically Altered Cell or Animal Models for ILK Function

The subject nucleic acids can be used to generate transgenic animals orsite specific gene modifications in cell lines. Transgenic animals maybe made through homologous recombination, where the normal ILK locus isaltered. Alternatively, a nucleic acid construct is randomly integratedinto the genome. Vectors for stable integration include plasmids,retroviruses and other animal viruses, YACs, and the like.

The modified cells or animals are useful in the study of ILK functionand regulation. For example, a series of small deletions and/orsubstitutions may be made in the ILK gene to determine the role ofdifferent exons in integrin binding, kinase activity, oncogenesis,signal transduction, etc. Of interest are the use of ILK to constructtransgenic animal models for cancer, where expression of ILK isspecifically reduced or absent. Specific constructs of interest includeanti-sense ILK, which will block ILK expression and expression ofdominant negative ILK mutations. A detectable marker, such as lac Z maybe introduced into the ILK locus, where upregulation of ILK expressionwill result in an easily detected change in phenotype.

One may also provide for expression of the ILK gene or variants thereofin cells or tissues where it is not normally expressed or at abnormaltimes of development. By providing expression of ILK protein in cells inwhich it is not normally produced, one can induce changes in cellbehavior, e.g. through ILK mediated LEK-1 activity.

DNA constructs for homologous recombination will comprise at least aportion of the ILK gene with the desired genetic modification, and willinclude regions of homology to the target locus. The regions of homologymay include coding regions, or may utilize intron and/or genomicsequence. DNA constructs for random integration need not include regionsof homology to mediate recombination. Conveniently, markers for positiveand negative selection are included. Methods for generating cells havingtargeted gene modifications through homologous recombination are knownin the art. For various techniques for transfecting mammalian cells, seeKeown et al. (1990) Methods in Enzymology 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in culture. The transgenic animalsmay be any non-human mammal, such as laboratory animals, domesticanimals, etc. The transgenic animals may be used in functional studies,drug screening, etc., e.g. to determine the effect of a candidate drugon oncogenesis, down regulation of E-cadherin, up regulation of LEF-1,etc.

In vitro Models for ILK Function

The availability of a number of components in the integrin signalingpathway allows in vitro reconstruction of the pathway. Two or more ofthe components may be combined in vitro, and the behavior assessed interms of activation of transcription of specific target sequences;modification of protein components, e.g. proteolytic processing,phosphorylation, methylation, etc.; ability of different proteincomponents to bind to each other; utilization of GTP, etc. Thecomponents may be modified by sequence deletion, substitution, etc. todetermine the functional role of specific domains.

Drug screening may be performed using an in vitro model, a geneticallyaltered cell or animal, or purified ILK protein. One can identifyligands or substrates that bind to, modulate or mimic the action of ILK.Areas of investigation include the. development of treatments forhyper-proliferative disorders, e.g. cancer, restenosis, osteoarthritis,metastasis, etc.

Drug screening identifies agents that modulate ILK function. Agents thatmimic its function are predicted to activate the process of celldivision and growth. Conversely, agents that reverse ILK function mayinhibit transformation. Of particular interest are screening assays foragents that have a low toxicity for human cells. A wide variety ofassays may be used for this purpose, including labeled in vitroprotein-protein binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, and the like. Knowledge of the3-dimensional structure of ILK, derived from crystallization of purifiedrecombinant ILK protein, leads to the rational design of small drugsthat specifically inhibit ILK activity. These drugs may be directed atspecific domains of ILK, e.g. the kinase catalytic domain, ankyrinrepeat domains, pleckstrin homology domains, etc.

Among the agents of interest for drug screening are those that interferewith the binding of [Ptdins(3,4,5)P₃ ] to the PH domains of ILK andagents that inhibit the production of [Ptdlns(3,4,5)P₃] by PI(3) kinase.Such assays may monitor the ILK activity in the presence of[Ptdins(3,4,5)P₃] and a candidate agent, as described in the examples.

The term “agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of altering or mimicking thephysiological function of ILK. Generally a plurality of assay mixturesare run in parallel with different agent concentrations to obtain adifferential response to the various concentrations. Typically one ofthese concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles,. e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin, etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

Other assays of interest detect agents that mimic ILK function, suchintegrin binding, kinase activity, down regulation of E-cadherin, upregulation of LEF-1, binding properties, etc. For example, an expressionconstruct comprising a ILK gene may be introduced into a cell line underconditions that allow expression. The level of ILK activity isdetermined by a functional assay, as previously described. In onescreening assay, candidate agents are added, and the formation offibronectin matrix is detected. In another assay, the ability ofcandidate agents to enhance ILK function is determined. A functionalassay of interest detects the stimulation of cyclin D1 and/or cyclin Aexpression. Alternatively, candidate agents are added to a cell thatlacks functional ILK, and screened for the ability to reproduce ILK in afunctional assay.

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of cancer, etc. The compounds may also be used to enhance ILKfunction in wound healing, cell growth, etc. The inhibitory agents maybe administered in a variety of ways, orally, topically, parenterallye.g. subcutaneously, intraperitoneally, by viral infection,intravascularly, etc. Topical treatments are of particular interest.Depending upon the manner of introduction, the compounds may beformulated in a variety of ways. The concentration of therapeuticallyactive compound in the formulation may vary from about 0.1-10 wt %.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXAMPLE 1 Isolation of ILK cDNA

A partial cDNA, BIT-9, was isolated in a two-hybrid screen using a baitplasmid expressing the cytoplasmic domain of the, integrin subunit. TheBIT-9 insert was used to isolate clones from a human placental cDNAlibrary. A 1.8 kb clone, Plac5, was found to contain a high degree ofsimilarity to cDNAs encoding protein kinases (FIGS. 1a-b), andrecognized a widely expressed transcript of 1.8 kb in Northern blots(FIG. 1c). Deduced amino acid residues 186-451 from plac5 comprise adomain which is highly homologous with the catalytic domains of a largenumber of protein tyrosine and serine/threonine kinases (FIG. 1a).Residues 33-164 comprise four repeats of a motif originally identifiedin erythrocyte ankyrin (FIG. 1b), likely defining a domain involved inmediating additional protein-protein interactions. Affinity-purifiedanti-ILK antibodies (see methods described in Example 3) were used inWestern blot analyses of mammalian cell extracts, and detected aconserved protein of apparent Mr of 59 kDa (p59ILK, FIG. 1d).

FIG. 1 shows yeast two-hybrid cloning, characterization, and expressionof ILK. The full length ILK cDNA, Plac5, was isolated from a humanplacental library using the BIT-9 insert. Plac5 contains a 1509 bp openreading frame, with a presumptive initiator Met at nt 157, and an AAUAAAsignal 11 bp upstream of the polyadenylation site. In vitrotranscription and translation of Plac5 in rabbit reticulocyte lysatesyielded a protein of apparent Mr of 59 kDa. (b) A search of the PIRprotein database indicated homology with protein kinase subdomains I toXI, as identified by Hanks et al. We note sequence variations in the ILKsubdomains 1, VIb, and VII, relative to catalytic domains of knownprotein kinases. Subdomain I (residues 199-213), does not have thetypical GXGXXG motif, although this region in ILK is Gly-rich. Insubdomain VIb, Asp328 of ILK may compensate for the lack of theotherwise conserved Asp319. In subdomain VII, the DFG triplet is absentin ILK. The integrin binding site maps to amino acid residues 293-451(BIT-9). The ILK kinase domain is most highly related to the CTR1 kinaseof Arabidopsis thaliana (30% identity, P<10). The CTR1, B-raf, Yes andCsk kinase domains are aligned with Plac5. (c) Amino acid residues33-164 comprise four contiguous ankyrin repeats, as defined by Lux et.al. (d) BIT-9 was used to probe a blot of poly A+selected RNA (MTN I,Clontech) from various human tissues. (e) Whole cell lysates of mouse,rat and human cell lines (10 μg/lane) were analyzed by Western blottingwith the affinity-purified 92-2 antibody (see description of methods inExample 3). The ILK sequence data are available from GenBank underaccession number U40282.

In order to construct integrin “bait” plasmids, sequences encoding aminoacid residues 738-798 of the β₁, and residues 1022-1049 of the α₅integrin subunits were amplified from full-length cDNAs. The primersused were (a) 5′ amplification (SEQ ID NO:7)5′-GGCCGMTTCGCTGGAATTGTTCTTATTGGC-3′ and (b) 3′ amplification (SEQ IDNO:8) 5′-GGCCGGATCCTCATTTTCCCTCATACTTCGG-3′. PCR products weredirectionally cloned into pEG202, creating the LexA fusion baitplasmids, pEG202βhd 1INT and pEG202α₅ INT. pEG202β₁ INT and pEG202α₅ INTrepressed β-gal expression from the pJK101 reporter by 50-60% and70-75%, respectively, in host strain EGY48 (MATα, his3, trp1, ura3-52,LEU2::pLEU2-LexAop6, constructed by Erica Golemis, MassachussettsGeneral Hospital), confirming nuclear expression of the LexA fusions.Co-transformation of baits with the pSH18-34 reporter verified they weretranscriptionally inert. A galactose-inducible HeLa cDNA interactorlibrary was present on the TRP+vector, pJG4-5 (constructed by JenoGyuris, MGH). For the β₁ interaction trap, EGY48 was transformedsequentially with pEG202β₁ INT, pSH18-34 and pJG4-5, using the lithiumacetate protocol (transformation efficiency=5-6×10⁴/μg). 2×10⁶ primarytransformants were screened, of which forty-nine interacting clones wereconfirmed. The most frequent isolate (31/49) was a 700 bp insert, BIT-9.Retransformation of EGY48 with the BIT-9, pSH18-34, and pEG202β₁ INTplasmids resulted in strong β-galactosidase expression, confirming theinteraction. An identical screen, using pEG202α₅ INT as bait, resultedin the isolation of 16 positives, none of which were represented in theset of 49 β₁ interactors. Trapped inserts were used to screen WM35 humanmelanoma λgt10, and human placental λgt11 cDNA libraries, using-standard procedures. cDNA sequencing of multiple clones from eachlibrary was done using the dideoxy chain termination method (Sequenase2.0, U.S. Biochemical). For data analysis we used the Genetics ComputerGroup software package (version 7.0), and database searches wereaccomplished via the BLAST server at the National Center forBiotechnology Information.

EXAMPLE 2 Analysis of ILK In vitro

For analysis of kinase activity in vitro, a bacterially-expressed fusionprotein, GST-ILK²32, was SDS-PAGE band purified, and incubated with[γ-³2 P]ATP in the presence or absence of the exogenous substrate myelinbasic protein (FIG. 2). GST-ILK²32 autophosphorylated and labeled MBPefficiently in these assays (FIG. 2a). Anti-GST-ILK²32 (antibody 91-3)immunoprecipitates of PC3 cell lysates were incubated with [γ-³2 P]ATP,similar to experiments performed with purified recombinant GST-ILK²32.ILK immune complexes labeled a protein of apparent Mr of 59 kDa (FIG.2b), corresponding to p59_(ILK), as well as cellular proteins ofapparent Mr 32 kDa and 70 kDa, which may be endogenous ILK substrates(FIG. 2b). Cellular phosphoproteins (serine/threonine) of approximately32 kDa and 70 kDa, were also seen in β₁ integrin-specific immune complexkinase assays.

In ILK immune complex kinase assays a synthetic peptide representing theβ₃ cytoplasmic domain was phosphorylated, while a similar peptiderepresenting the β₃ cytoplasmic domain was not detectably labeled byp59_(ILK). The β₁ peptide selectively inhibited autophosphorylation ofILK in these reactions (FIG. 2b), further indicating a differentialinteraction of the peptides with ILK. The results demonstratingphosphorylation of synthetic β peptides by endogenous ILK are identicalto those seen with recombinant GST-ILK²32, and indicate the potentialsubstrate preference of ILK for the β₁ cytoplasmic tail. This does not,however, necessarily rule out an interaction between ILK and the β₃integrin cytoplasmic domain. Phosphoamino acid analyses of labeledp59_(ILK) and MBP from the immune complex kinase assays detected onlyphosphoserine in both substrates (FIG. 2c), as was the case forphosphorylation of these substrates by. GST-ILK²32. The β₁ peptide waslabeled on serine and threonine residues, with approximately equalstoichiometry (FIG. 2). As a control, anti-FAK immune complexes from thesame Iysates were analyzed for phosphorylation of MBP, andphosphotyrosine was readily detected.

FIG. 2 shows in vitro and immune-complex kinase assays. a, In vitrokinase reactions containing 2 μg of gel-purified GST-ILK132, with andwithout 5 μg of myelin basic protein (MBP, Upstate Biotechnologies,Inc.), were analyzed by 10% SDS-PAGE. b, Immune complexes were generatedfrom PC3 whole cell lysates, using affinity-purified 91-3 antibody.Complexes were assayed for kinase activity, with and without addition of5 μg/reaction of synthetic peptides, representing β₁ or β₃ integrincytoplasmic domains or MBP. Products were analyzed by 15% SDS-PAGE (kDamarkers at left), and migration of peptides confirmed by Coomassie Bluestaining. c, ³2 P-labeled products from the anti-ILK immune complexkinase reactions shown in b, were isolated and analyzed forphosphoamino- acid content. Anti-FAK immune complex kinase assaysdemonstrated phosphotyrosine on MBP.

Protein kinase assays were performed in 50 μl kinase reaction buffer (50mM HEPES pH 7.0, 10 mM MnC₂, 10 mM MgCl₂, 2 mM NaF, 1 mM Na₃ VO₄),containing 10 μCi [γ-³2 P]ATP. Reactions were incubated at 30° C. for 20min, and stopped by the addition of SDS-PAGE sample buffer. For assay ofrecombinant ILK activity, GST-ILK²32 was adsorbed from bacterial lysatesonto glutathione-agarose beads, or GST-ILK²32 was band-purified from 10%SDS-PAGE gels. For immune complex kinase assays, affinity-purified 91-3anti-ILK antibody (FIG. 3) was used to generate immunoprecipitates fromNP-40 lysates (150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) sodiumdeoxycholate, 50 mM HEPES pH 7.5, 1 μg/ml each leupeptin and aprotinin,50 μg/ml phenyl-methylsulfonyl flouride) of PC3 cells. Kinase reactionproducts were resolved on 10-15% SDS-PAGE gels, transferred to PVDF, andphosphoamino acid analysis performed according to a published protocol.

EXAMPLE 3 Association of ILK and β integrin in Mammalian Cells

Immunofluorescence experiments indicated that ILK and β integrinco-localize in focal plaques. In order to test further for thisassociation in intact mammalian cells, we performedco-immunoprecipitation assays in lysates of PC3 cells, in which integrinexpression has been well-characterized. PC3 cell lysates wereimmunoprecipitated with specific anti-integrin antibodies, and immunecomplexes analyzed by Western blotting with the anti-ILK antibody, 92-2.The specificities of the anti-ILK antibodies were tested byimmunoprecipitation and Western blotting (FIGS. 3a, b). We detectedp59_(ILK) in immune complexes obtained with anti-fibronectin receptor(FNR, α₅/α₃ β integrin), and anti-vitronectin receptor (VNR, α.sub.v β₃/β₅ integrin) antibodies, but not in those obtained with non-immuneserum (FIG. 3c). Three anti-β₁ monoclonal antibodies alsoco-precipitated p59_(ILK) from PC3 lysates, confirming the β integrinspecificity of p59_(ILK) interaction (FIG. 3d). The detection ofp59_(ILK) in anti-VNR immune complexes suggests that ILK may alsointeract with the β₃ and/or β₅ integrin subunit(s).

FIG. 3 shows that antibodies to GST-ILK²32 recognize p59_(ILK) inintegrin co-immunoprecipitations. a, Unfractionated polyclonal anti-ILKsera 91-3 (shown) and 92-2 specifically recognize a ³5 S-methionine,metabolically-labeled cellular protein, of apparent Mr of 59 kDa. Afluorograph is shown (En³ Hance, NEN). b, Affinity-purified 92-2antibody was adsorbed with 165 μg of agarose-coupled GST-ILK²32, oragarose-GST, which preparations were used in parallel Western blotscontaining 10 μg/lane of whole cell lysates of PC3 cells, JurkatT-lymphoblasts, or the 60 kDa GST-ILK²32. c, Polyclonal anti-integrinantibodies, specific for the fibronectin and vitronectin receptors, wereused to precipitate surface-biotinylated integrins from PC3 cells, andimmune complexes were then analyzed for the presence of p59_(ILK), byWestern blotting with affinity-purified, biotin-labeled 92-2 antibody.This result is representative of six independent experiments. d, Anti-β₁monoclonal antibodies were used in co-precipitation analyses of NP-40lysates of PC3: lane 1, A.sub.II B₂; lane 2, anti-CD29; lane 3, 3S3.Western blotting of anti-β₁ immune complexes with affinity-purified,biotinylated 92-2 antibody (left). This blot was stripped and reprobedwith the same concentration of biotinylated 92-2, adsorbed against anexcess of GST-ILK²32 beads (right). We observe co-precipitation ofp59_(ILK) using a panel of 11 anti-β₁ monoclonals, but not with ananti-CD44 monoclonal antibody. The migration of p59_(ILK) was confirmedin parallel lanes containing PC3 whole cell NP-40 lysates. Markers atleft, in kDa.

Amino acid residues 132-451 of ILK were expressed as a GST fusionprotein, in E. coli. Recombinant GST-ILK²32 protein was purified andused to inject two rabbits. The resulting antisera, 91-3 and 92-2(raised by Research Genetics, Inc.), were affinity-purified over acolumn of CNBr-Sepharose coupled GST-ILK²32. PC3 cells weremetabolically labeled with 100 μCi/ml [³5 S]methionine [³5 S]cysteine([³5 S] ProMix, 1000 Ci/mmol, Amersham), for 18 hours incysteine/methionine-free MEM. For co-immunoprecipitation experiments PC3cells were surface-labeled with sulfo-NHS-biotin (Pierce Chemicals),prior to lysis in NP-40 buffer. Polyclonal anti-fibronectin receptor(anti-FNR, Telios A 108), and anti-vitronectin receptor (anti-VNR,Telios A 109) antibodies were purchased from Gibco/BRL. 1-2 mg of NP-40lysate was incubated at 40° C., with 2-3 μl/ml anti-FNR or anti-VNRantiserum, or 2 μg/ml of the anti-β₁ monoclonal antibodies A.sub.II B₂(C. Damsky, UC, San Francisco), anti -CD29 (Upstate Biotechnology,Inc.), and 3S3 (J. Wilkins, U Manitoba). Lysates were pre-cleared andimmune complexes collected with Protein A-Sepharose. For Westernblotting, RIPA lysates or immune complexes were subjected to 7.5% or 10%SDS-PAGE, and proteins then electrophoretically transferred topolyvinylidene fluoride membranes (Immobilon-P, Millipore). Membraneswere blocked in 5% non-fat milk/Tris-buffered saline Tween-20, andincubated with 0.5 μg/ml affinity purified antibodies. Horseradishperoxidase-coupled goat anti-rabbit IgG was used in secondaryincubations, followed by detection of reactive bands by enhancedchemiluminescence (ECL, Amersham). For blotting without use of secondaryantibody (FIG. 3), affinity-purified 92-2 antibody was labeled withBiotin Hydrazide (Immunopure, Pierce Chemicals), according to themanufacturer's protocol, with visualization by peroxidase-conjugatedstreptavidin (Jackson ImmunoResearch Laboratories) and ECL. Forre-probing, membranes were stripped according to manufacturer'sinstructions.

EXAMPLE 4 Overexpression of ILK Provides Growth Advantage

The fibronectin-dependent regulation of ILK kinase activity was tested.Plating of rat intestinal epithelial cells, IEC-18 on fibronectinreduced ILK phosphorylation of MBP in immune complex kinase assays,relative to cells plated on plastic, or kept in suspension (FIG. 4a).This fibronectin-dependent reduction of ILK activity was abrogated inIEC-18 cells expressing an activated H-ras allele, indicating that rastransformation disrupts ECM regulation of ILK activity in these cells.An expression vector containing the full-length ILK cDNA, PCMV-ILK, wasstably transfected into IEC-18 cells. Twelve stable clones each, ofpCMV-ILK and vector control transfectants, were selected andcharacterized for p59_(ILK) expression levels. Two representativeoverexpressing subclones, ILK13-A1a3 and -A4a are illustrated (FIG. 4b).Overexpression of p59_(ILK) disrupted the epithelial morphology ofIEC-18 cells. ILK13 clones were more refractile, and grew on LN, FN andVN with a stellate morphology, in marked contrast to the typical,‘cobble-stone’ morphology of the parental and ILK14 cells (FIG. 4c). Weplated the ILK13-A1a3 and -A4a subclones, the control transfectants,ILK14-A2C3 and -A2C6, and IEC-18 cells, on varying concentrations of theintegrin substrates, laminin (LN), fibronectin (FN) and vitronectin(VN). Adhesion of the ILK14 and IEC-18 cells was equivalent, whereasthat of the overexpressing subclones was significantly reduced, on allthese substrates (FIG. 4d). Immunoprecipitation analysis indicated thatcell surface integrin expression was unaffected. The effect of p59_(ILK)overexpression on anchorage-independent growth was examined by assayingthe colony forming ability of ILK transfectants in soft agarose. Inmarked contrast to IEC-18 and transfectant controls, four independentp59_(ILK) overexpressing subclones, ILK13-A4a, A1a3, A4d3 and A4C12,formed colonies in these assays (FIG. 4e). The proliferative rates ofall of these clones on tissue culture plastic were equivalent to controlrates.

FIG. 4 shows the modulation of ILK kinase activity by ECM components. a,ILK phosphorylation of MBP was assayed in ILK immune complexes, fromlysates of IEC-18 intestinal epithelial cells which were harvested fromtissue culture plastic and either kept in suspension, or replated onfibronectin, for 1 hour. A H-ras-transformed variant of IEC-18, Ras37(transfected with Rasval12 in pRC/CMV vector), was assayed in parallel.The band shown is MBP. b, Expression levels of p59_(ILK) in tworepresentative clones of IEC-18 cells, transfected with an. ILKexpression construct (ILK13), two vector control clones (ILK14), and theparental IEC-18 cells are presented. The indicated amounts (μg/lane) ofwhole cell RIPA lysates were run out on 10% SDS-PAGE gels, and p59_(ILK)expression analyzed by Western blotting with affinity-purified 92-2antibody. c, Representative p59_(ILK) overexpressing clone ILK13-A4a,vector control clone ILK14-A2C3, and parental IEC-18 cells were platedon the ECM substrates LN, FN and VN for 1 hour, then fixed, stained withtoluidine blue and photographed (40×mag). d, Adhesion of the ILKoverexpressing clones to LN, FN and VN was quantified. Key: IEC-18(black), ILK14-A2C6 (white), ILK13-A1a3. (dark grey), ILK13-A4a (lightgrey). Results are presented for 10 μg/ml substrate, and are expressedas % adhesion (+/−s. d.) relative to IEC-18, for each substrate. Theserial concentrations of ECM showed similar reductions in adhesion ofthe ILK13 subclones, and ILK14-A2C3 adhesion was identical to that ofILK14-A2C6, on all three substrates. Immunoprecipitation ofsurface-biotinylated IEC-18, ILK13, and ILK14 subclones, with theanti-FNR and anti-VNR sera, confirmed there was no change in expressionof α₅/α₃β₁ and α.sub.v β₃/β₅ integrin subunits in the p59_(ILK)overexpressors. Data are representative of two independent experiments.e, Four ILK13, p59_(ILK) overexpressing clones were plated in softagarose, and assayed for colony growth after three (experiment 1) andtwo (experiment 2) weeks. Parent and vector control transfectants werealso assayed, and the ras val12 transformed clone, Ras-37, was used as apositive control. Bars represent the mean of duplicate determinations.Maximum colonies in IEC-18 and ILK14 cells was 1/field.

The rat intestinal epithelial cell line IEC-18, and a variant of thisline transfected with an activated H-rasval12 allele, expressed frompRC/CMV, were grown on tissue culture plastic in 5% serum-containingmedium, washed three times in minimum essential medium (MEM), andharvested with 5 mM EDTA. These were resuspended in 2.5 mg/ml BSA inMEM, and either kept in suspension, or plated on 10 μg/mlfibronectin-coated plates, for 1 hour at 37° C. NP-40 lysates (300 μg)of these cells were immunoprecipitated with affinity-purified 91-3, andimmune complex kinase assays (MBP substrate) performed, as describedabove. IEC-18 were transfected with the expression vector pRC/CMV,containing Plac5 in the forward orientation relative to the CMVpromotor. Stable clones were selected in G418, and subcloned through tworounds of limiting dilution. In all, twelve each of ILK and vectorcontrol transfectant subclones were isolated. Protein concentrationswere determined using the Bradford reagent (Bio-Rad). Two p59_(ILK)overexpressors, ILK13-A1a3 and ILK13-A4a, and two vector transfectantcontrols, ILK14-A2C3 and -A2C6, were analyzed for effects of ILKoverexpression on cell adhesion to ECM substrates. Adhesion wasquantified according to published methods. For colony formation assays3×10⁵ cells were plated in 35 mm wells, in 0.3% agarose, as describedpreviously. Ras-37 were plated at 2×10³/well. Colonies were counted andscored per field (d=1 cm) in duplicate wells, and defined as a minimumaggregate of 50 cells.

These results demonstrate that p59_(ILK) overexpression in the IECepithelial cells provides a growth advantage, in the absence ofproliferative signals normally provided by adhesion.

The transduction of extracellular matrix signals through integrinsinfluences intracellular (‘outside-in’) and extracellular (‘inside-out’)functions, both of which appear to require interaction of integrincytoplasmic domains with cellular proteins. The association of ILK withβ₁ integrin subunits, and specific regulation of its kinase activity byadhesion to fibronectin, suggests that p59_(ILK) is a mediator ofintegrin signaling. Thus the ankyrin repeat motif likely represents aprotein interaction module specifying interactions of ILK withdownstream, cytoplasmic or cytoskeletal proteins. Reduced ECM adhesionby the p59_(ILK) overexpressing cells is consistent with our observationof adhesion-dependent inhibition of ILK activity, and suggests thatp59_(ILK) plays a role in inside-out integrin signaling. Furthermore thep59_(ILK) -induced, anchorage-independent growth of epithelial cellsindicates a role for ILK in mediating intracellular signal transductionby integrins.

EXAMPLE 5 The Effect of Anti-ILK on Cell Migration

The role of ILK in cell motility has important implications for normalphysiological processes such as inflammation and wound healing, as wellas pathological conditions involving tumour invasiveness and metastatictumour spread, or osteoporosis (bone is essentially an extracellularmatrix secreted by osteoblast, or bone-forming cells, and thisdeposition can be modulated by integrin expression levels and function).Cell motility is a dynamic process, which is dependent on integrin-ECMinteractions. The “on-off” switch function of protein kinases providesan ideal mechanism for the dynamic regulation of integrin affinitystates for ECM substrates. The effect on cell migration ofmicroinjecting highly specific anti-ILK antibodies (thereby inhibitingILK function) into the cell's cytoplasm is assayed. These effects areassayed in endothelial cells plated on solid substrata, and are extendedto include studies on cell migration through three-dimensional gelscomposed of ECM proteins.

EXAMPLE 6 Anti-Sense Oligonucleotides to Inhibit ILK Activity

The sequence of ILK cDNA provides information for the design andgeneration of synthetic oligonucleotides for “anti-sense” inhibition ofILK activity. This term derives from the strategy of employing a reversecomplement of the coding, or sense strand of a specific messenger RNA,known as an anti-sense oligonucleotide (AO). By binding to itscomplementary mRNA, the AO inhibits translation of that mRNA intoprotein, thereby preventing normal protein accumulation in the cell. ILKAO derived from the ILK mRNA sequence closest to the presumptivetranslational start site, as defined in FIG. 1, is tested, as this ispredicted to provide the most successful reagent.

Regardless of the actual chemistry used to construct the AO, ormodifications to an anti-ILK AO to improve its efficiency, the cDNAsequence of ILK provides the information for derivation of a specificAO. The cDNA sequence of ILK is also used to design oligonucleotidereagents, known as degenerate primers (due to the degeneracy of thegenetic code), for use in polymerase chain reaction (PCR)-based screensfor cDNAs structurally related to ILK. Similarly, the ILK CDNA is usedto screen for related genes in a more conventional screen of genomic orcDNA libraries, by employing less stringent (i.e. milder) hybridizationconditions during screening. In this way, distinct cDNA or DNA sequencessignificantly related to ILK (>50% nucleotide identity) can be isolated,and a family of ILK-related kinases identified in a non-random fashion.

EXAMPLE 7 Mapping of ILK Chromosomal Locus to Assess Imprinted Copies ofGene

High resolution mapping of the ILK chromosomal locus through fluorescentin situ hybridization (FISH) to metaphase (i.e. separated andidentifiable) human chromosomes has placed the ILK gene on chromosome 11p15. FISH is known to those skilled in the art. High resolution mappinguses known marker genes in this region. Certain genes (e.g. insulin-likegrowth factor 2, IGF2) in the 11p15 region have been shown to beimprinted (i.e. preferentially expressed from either the maternally orpaternally-derived chromosomes). This imprinting effectively provides afunctional deletion or “knock-out” of one of the two inherited copies ofa gene. Thus, mutation of the non-imprinted allele (copy) has a moreprofound outcome, since no compensatory activity is available from theimprinted allele. Also, 11p15 has been identified as a region subject toloss-of-heterozygosity, or LOH, in a subset of breast tumour patients.LOH results in the loss of one allele, for example by gene deletion, andis a mechanism underlying the contribution of a number of tumorsuppressor genes to the development of various cancers (e.g. BRCA1 inbreast, DCC in colon carcinoma, and RB1 in retinoblastoma). Thus ILKcDNA sequence is used to develop DNA reagents for the diagnosis andprognostic indications of a significant subset of breast cancers, andthese reagents contribute to the molecular classification of suchtumors. As mentioned above, the gene(s) on 11 p15 contributing to someinherited cases of long QT syndrome are identified, and the candidacy ofILK as a causative gene for this cardiac condition, are evaluated bylooking for alterations in ILK gene structure in families where 11p15associations have been made.

EXAMPLE 8 Induction of in vivo Tumorigenesis by Overexpression of ILK

Overexpression of ILK down-regulates E-cadherin which is an importantepithelial cell adhesion molecule mediating cell-cellcommuncation/interaction. The loss of E-cadherin induced byoverexpression of ILK in epithelial cells suggests that ILK may promotetumorigenicity in vivo. To test this, we injected cells expressingvarying levels of ILK into athymic nude mice subcutaneously. Mice wereinoculated subcutaneously with the cells expressing high (ILK13-A1a3 andA4a) or low (IEC-18 and ILK14-A2C3) levels of ILK (10⁷ cells/mouse inPBS). The mice were monitored for tumor formation at the site ofinoculation after three weeks. Tumors arose within three weeks in 50% to100% of the mice injected with the ILK13 cells (10.sup.7 cells/mouse)that overexpress ILK, whereas no tumors were detected in the mice thatwere injected with the same number of the IEC-18 or ILK14 cellsexpressing lower levels of ILK (Table 1). Thus, overexpression of ILK inthese epithelial cells promotes tumor formation in vivo.

TABLE 1 Tumorigenicity of ILK Overexpressing IEC-18 Cells Cell Number ofMice with Line Tumors at 3 weeks IEC-18 0/6 ILK-A2C3 0/6 ILK13-A1a3 6/6ILK13-A4a 3/6

EXAMPLE 9 Increased Expression of ILK in Human Breast Carcinoma

The expression of Integrin Linked Kinase in human breast carcinomas wasdetermined by immunohistochemical staining of paraffin embedded sectionsfrom human breast cancer biopsies. Affinity purified anti-ILK polyclonalantibody was used followed by conjugated secondary antibody. Thepositive staining observed was completely abolished by absorption of theantibody to ILK-coupled sepharose beads. A total of 30 samples have beenexamined so far. In every case ILK expression levels are markedlyelevated in tumor tissue compared to normal ducts and lobules. FIG. 5Ashows a normal region showing well formed ducts with a single layer ofepithelial cells. ILK staining is most prominent in epithelial cells.The stroma appears negative. FIG. 5B shows ductal carcinoma in situ(DCIS). Multiple cell layers are present with markedly elevated ILKstaining in the tumor cells. Invasive carcinoma is depicted in FIGS. 5Cand 5D. There is markedly elevated expression of ILK compared to thenormal tissue shown in FIG. 5A.

EXAMPLE 10 Regulation of LEF-1 Expression and Complex Formation

Overexpression of ILK results in a downregulation of E-cadherinexpression, formation of a complex between β-catenin and the HMGtranscription factor, LEF-1, translocation of β-catenin to the nucleus,and transcriptional activation by this LEF-1/β-catenin complex. LEF-1protein expression is rapidly modulated by cell detachment from theextracellular matrix, and that LEF-1 protein levels are constitutivelyupregulated upon ILK overexpression. These effects are specific for ILK,since transformation by activated H-ras or v-src oncogenes do not resultin the activation of LEF-1/β-catenin. The results demonstrate that theoncogenic properties of ILK involve activation of the LEF-1/β-cateninsignaling pathway via elevation of LEF-1 expression.

Overexpression of ILK in rat intestinal epithelial cells.(IEC-18)induces a loss of epithelial morphology, characterized by a disruptionof cell-cell adhesion and the acquisition of a fibroblastic morphologythat includes enhanced fibronectin matrix assembly. This alteredmorphology is accompanied by the ability of the cells to progressthrough the cell cycle in an anchorage-independent manner and to formtumors in nude mice. To determine whether the loss of cell-cell adhesionwas accompanied by an increased invasive phenotype, the invasiveness ofIEC-18 parental cells and ILK-overexpressing (ILK-13) cells was testedin a collagen gel invasiveness assay. The data is shown in Table 2.

The ILK-13 cells are much more invasive than the parental and controltransfected (ILK-14) cells that have been transfected with an ILKanti-sense cDNA construct. Collagen-gel invasion by epithelial cells isnormally associated with an epithelial to mesenchymal transformationcharacterized by the down regulation of E-cadherin expression. Notably,the expression of E-cadherin protein is completely lost in ILKoverexpressing cells (ILK-13), but is maintained in control transfectedcells, reduced in IEC-18 cells transfected with activated H-ras cDNA,and greatly reduced in v-src transformed cells. In contrast, thesteady-state levels of the expression of the intracellular E-cadherinbinding protein, β-catenin, is unchanged by ILK overexpression and issimilar in all IEC cell transfectants.

The subcellular localization of β-catenin was examined in these cells.In sharp contrast to the localization of β-catenin at the plasmamembrane and at cell-cell adhesion sites in the parental IEC-18 andcontrol transfected cell clones (A2c3 and A2c6), β-catenin is localizedentirely in the nuclei of ILK overexpressing ILK-13 clones (A4a, A1a3).This ILK-induced nuclear localization of β-catenin is dependent on anactive kinase, since overexpression of a kinase-deficient ILK (E359K)did not induce nuclear translocation of β-catenin which remainslocalized largely to the plasma membrane; Likewise, overexpression ofkinase-deficient ILK also did not result in a loss of E-cadherinexpression. The translocation of β-catenin to the nucleus is a specificproperty of ILK, since in IEC-18 cells transfected with activated H-rasor v-src oncogenes, β-catenin is not translocated to the nucleus, but iseither localized to the plasma membrane or is expressed diffusely in thecytoplasm. Although these oncogenes also disrupt the epithelialmorphology of IEC-18 cells and result in the downregulation ofE-cadherin expression, the translocation of β-catenin to the nucleus isa property unique to ILK expression, suggesting that loss of E-cadherinexpression and β-catenin nuclear translocation may be regulateddifferentially. Overexpression of ILK in mouse mammary epithelial cellsalso results in similar alterations in the phenotypic propertiesdescribed above for the IEC-18 cells.

Translocation of β-catenin to the nucleus can be induced by theactivation of the Wnt signaling pathway, which initially results in anelevation of free cytosolic β-catenin due to decreased degradation.Alternatively, loss of expression or mutations in the tumor suppressorprotein APC and certain mutations in the β-catenin gene lead tocytosolic β-catenin stabilization and nuclear translocation. The nucleartranslocation of β-catenin is associated with complex formation betweenβ-catenin and members of the HMG transcription factors, LEF-1/TCF whichthen activate (or silence) transcription of target genes. Since thesteady state levels of β-catenin were not changed by ILK overexpression“uncomplexed” β-catenin levels were measured, as determined by bindingto a cytoplasmic domain peptide of E-cadherin. “Uncomplexed ” pools ofβ-catenin in ILK overexpressing clones were found to be low andunaltered compared to IEC-18 cells or control ILK 14 clones. Thisindicates that most β-catenin is likely complexed with nuclearcomponents such as transcription factors and DNA. In contrast, freeP-catenin pools in Ras and Src transformed cells were high consistentwith decreased E-cadherin expression and indicating disruption ofE-cadherin-β-catenin interaction. However, the increased free pools ofβ-catenin did not result in nuclear translocation of β-catenin.

The expression levels of LEF-1, a member of the family of HMGtranscription factors that bind β-catenin, were measured. The expressionof LEF-1 is dramatically higher in six independent ILK expressing ILK-13cell clones as compared with six independent control transfected ILK-14clones, as well as 2 activated H-ras transfected and v-src transfectedIEC-1 8 clones. E-cadherin expression is lost in all 6 ILK-13 celllines. Transient induction of ILK expression using an ecdysone inducibleILK construct also resulted in an increase of LEF-1 expression. Asexpected, the increased levels of LEF-1 and the nuclear translocation ofβ-catenin are associated with enhanced complex formation between LEF-1and β-catenin in the ILK overexpressing cells.

LEF-1 is a transcription factor that is by itself, unable to stimulatetranscription from multimerized binding sites, however in associationwith β-catenin, LEF1/TCF proteins can augment promoter activity frommultimerized binding sites. Transcriptional activation from aTCF/β-catenin responsive promoter construct was examined inILK-overexpressing cells and control kinase-deficient ILK expressingcells. High promoter activity was observed in ILK-overexpressing cellsand the extent of transcriptional activation was reduced with promoterconstructs containing mutations in the multimerized LEF-1/TCF bindingsites. Moreover, nuclear extracts were analyzed from ILK-overexpressingcell clones and from cell clones transfected with an anti-sense orkinase-deficient ILK cDNA to identify proteins that bind the LEF/TCFbinding site. The abundance of a nuclear factor in ILK-overexpressingcells that displays the same binding site specificity, immunoreactivityand electrophoretic mobility as LEF-1, was found to be markedly enhancedrelative to the unrelated DNA-binding protein Oct-1.

ILK binds to the cytoplasmic domain of β₁ and α₃ integrin subunits, andits kinase activity is downregulated upon cell adhesion to extracellularmatrix (ECM) proteins. Overexpression of constitutively activated ILKovercomes this regulation of ILK activity by integrin occupation andresults in decreased cell adhesion to ECM-protein. Cell adhesion to ECMsuppresses LEF-1 expression, which is rapidly, but transiently, elevatedupon cell detachment in ILK-14 and ILK13 cells. However in ILKoverexpressing ILK-13 cells the elevation in LEF-1 levels are morerobust and are maintained at high levels for as long as 16 hours insuspension. Furthermore, LEF-1 levels are also higher in adherent ILK-13cells compared to ILK-14 cells.

These data indicate that ILK overexpression overcomes the regulation ofLEF-1 expression by adhesion-deadhesion, and that the maintenance ofconstitutively high levels of LEF-1 result in enhanced complex formationbetween LEF-1 and β-catenin, translocation of β-catenin to the nucleus,and transcriptional activation of responsive genes. Since TCF/β-cateninhas been shown to induce transcription of genes encoding homeoboxproteins that regulate mesenchymal genes eg. Siamois in Drosophila, thispathway is likely to mediate the observed epithelial to mesenchymaltransformation, as well as the oncogenic properties of ILK in theseintestinal epithelial cells, since constitutive activation ofTCF/β-catenin is oncogenic in human colon carcinomas. The data presentedhere also suggest a connection between the expression of E-cadherin andthe signaling properties of β-catenin in mesenchymal induction in ILKtransformed cells, in agreement with the work of others that E-cadherincan antagonize β-catenin signaling, although the loss of E-cadherinexpression does not always correlate with nuclear β-catenintranslocation e.g. in the v-src transformed cells.

An additional pathway is demonstrated to that by activated Wnt-1 leadingto increased LEF-1/β-catenin complex formation and transcriptionalactivation. These data also corroborate previous work showing thatoverexpression of LEF-1 can work independently of Wnt to enhanceLEF-1β-catenin complex induced transcription. Here it is shown that incontrast to the effects of Wnt-1, activated ILK can dramatically inducethe formation and nuclear translocation of LEF-1/β-catenin complexeswithout a corresponding increase in the free pool of β-catenin. ThisILK-regulated pathway may be modulated via cell adhesion to ECM, but canbe constitutively activated by ILK overexpression.

Methods

Cells and cell culture. IEC-18 rat intestinal epithelial cells werestably transfected with a mammalian vector incorporating ILK to produceclones overexpressing wt ILK in the sense orientation (ILK-13) orantisense orientation (ILK-14), or to produce a kinase-deficient form ofILK (IEC-18GH31 RH) described below. IEC-18 cells were also stablytransfected to overexpress H-ras (Ras 33, Ras 37) (Buick et al. (1987)Exp. Cell. Res. 170:300-309), and v-src (Src2, Src4) (Filmus et al.(1988) Mol. Cell. Biol. 8:4243-4249). Cells were grown in d-MEMcontaining 5% FCS, 2 mm L-glutamine, glucose (3.6 mg/ml), insulin (10ug/ml); and G418 (40 ug/ml) was added to transfected cells to maintainselection pressure.

Site directed mutagenesis of ILK kinase domain. Mutations wereintroduced into wt ILK-cDNA with the Promega Altered Sites II System(Promega, Madison Wis.). Mutant oligomers (with the altered nucleotideunderlined) were used to change lysine at position 220 to an arginine(K220R, (SEQ ID NO:9) 5′ CCTTCAGCACCCTCACGACAATGTCATTGCCC 3′) andglutamic acid at position 359 to lysine (E359K, (SEQ ID NO: 10) 5′CTGCAGAGCTTTGGGGGCTACCCAGGCAGGTG 3′). Mutant clones were confirmed bydideoxy sequencing and subcloned into pGEX4T-1 GST fusion vector(Pharmacia, Piscataway N.J.) to express GST-ILK in E. coli (BL21-DE3)and into pcDNA3 (Invitrogen, San Diego, Calif.) to stably transfectkinase deficient ILK into the IEC-18 cell line (IEC-18GH31RH containingthe E359K mutation).

Inducible expression of ILK. Full length wt ILK CDNA (1.8 Kb) wassubcloned into the Ecdysone-inducible expression vector pIND(Invitrogen, San Diego, Calif.) and 10 ug were transientlyco-transfected with 10 ug of the complementary regulator vector pVgRXRinto subconfluent cells growing in 6 well plates with 20 ul ofLipofectin (Gibro-BRL, Gaithersburg, Md.). ILK expression was induced 6hrs later with the addition of 1 uM muristerone A.

Western blotting and immunoprecipitation. Cells were lysed for 10minutes on ice in NP-40 lysis buffer (1% NP-40, 50 mM Hepes, pH 7.4, 150mM NaCl, 2 mM EDTA, 2 mM PMSF, 1 mM Na-o-vanadate, 1 mM NaF, 10 ug/mlaprotinin, 10 ug/ml leupeptin). Extracts were centrifuged with theresulting supernatants being the cell lysate used in assays. Lysateswere electrophoresed through SDS-PAGE and transferred to lmmobilon-Pmembranes (Millipore, Bedford; Md.). Antibodies used to probe Westernblots were: rabbit anti-ILK, monoclonal anti-E-cadherin and monoclonalanti-β-catenin (Transduction Labs, Lexington, Ky.), and rabbitanti-LEF-1 (Travis et al. (1991) Genes & Development 5:880-894). Bandswere visualized with ECL chemiluminescent substrate (Amersham,Buckinghamshire, England). For immunoprecipitation, NP-40 lysates wererotated with primary antibody ON at 4° C., then rotated with ProteinG-Sepharose (Pharmacia, Uppsala, Sweden) for 2 hrs at RT. Beads werepelleted, boiled in electrophoresis sample buffer (non-reducing),centrifuged and supernatants were electrophoresed. Proteinconcentrations were determined by the Bradford assay (Bio-Rad, Hercules,Calif.).

Invasion assay. Confluent cells were trypsinized and 7.5×10⁴ cells in1.5 ml of complete medium were seeded onto 1.5 ml of a three dimensionalcollagen gel in a 35 mm tissue culture dish (Montesano et al. (1985)Cell 42:469-477). Upon reaching confluence (3 days), the cultures wereincubated for a further 4 days, then fixed in situ with 2.5%glutaraldehyde in 100 mM cacodylate buffer (pH 7.4), and photographed atdifferent planes of focus. Invasion was quantitated by counting thenumber of cells which had migrated below the surface of the collagengel. Five randomly selected fields measuring 1.0 mm×1.4 mm werephotographed at a single level beneath the surface monolayer using a 10×phase contract objective.

Indirect immunofluorescence. Cells were grown on cover slips, washedwith PBS, fixed in 4% paraformaldehyde in PBS for 12 minutes, washedwith PBS, permeabilized in 0.1% Triton X-100 in PBS for 10 minutes,blocked with 4% BSA in PBS for 30 minutes at RT, incubated with rabbitanti-β-catenin (Hulsken et al. (1994) J. Cell Biol. 127:2061-2069)diluted 1:400 in 0.1% Triton X-100 for 60 minutes at 37° C., Washed withPBS, incubated with rhodamine conjugated goat anti-rabbit IgG (JacksonImmunoResearch, West Grove, Pa.) diluted 1:50 in 0.1% Triton X-100 for30 minutes at 37° C., washed with PBS, then mounted onto a slide withSlow-Fade Antifade (Molecular Probes Inc., Eugene, Oreg.). Cells wereviewed at 100 fold magnification using a Zeiss Axiovert 135 fluorescencemicroscope.

Reportergene assay. Cells were transiently transfected with 0.3 ug of aluciferase reporter gene construct containing a series of optimal ormutated LEF-1/TCF binding sites (Korinek et al. (1997) Science275:1784-1787), along with 0.05 ug of a CAT gene construct containing aribosomal promoter (Hariharan et al. (1989) Genes & Development3:1789-1800) to control for transfection efficiency. Extracts wereprepared and assayed 48 hours after transfection.

Electrophoretic mobilty shift assay. Twenty μg of nuclear extract wereincubated with 1 fmole of ³2 P-labeled duplex oligonucleotide probespecific for LEF-1, in 20 μl of binding buffer containing 200 ngpoly[d(I-C)], 400 ng salmon sperm DNA, and electrophoresed through a 5%native polyacrylamide gel (Travis et al. (1991) Genes and Development5:880-894). For DNA competition, an 800-fold molar excess ofoligonucleotide containing a specific LEF-1 binding site or anon-specific EBF-binding site (Hagman et al. (1991) EMBO J.10:3409-3417) was included in the DNA-binding reaction. For antibodyaddition, 1 ul of polyclonal anti-LEF-1 antibody or 1 ul of monoclonalanti-β-catenin antibody (Transduction Labs, Lexington, Ky.) were used.

TABLE 2 INVASION OF COLLAGEN GELS Cell Line Invading cells/field IEC-18   10 +/− 0.87 ILK14/A2c  67.8 +/− 1.32 ILK13/A1a 326.73 +/− 2.61ILK-13/A4a  83.6 +/− 4.68

After seeding 7.5×10⁴ cells, the number of invading cells in 5photographic fields from 3 separate experiments (total of 15 fields/cellline) were counted. Results are given as the mean number of invadingcells +/−SEM. *p<<0.01 between ILK13/A1a3 compared to IEC-18 and ILK-14cells (Students unpaired t=test).

EXAMPLE 11 Regulation of Fibronectin Matrix Assembly, E-cadherinExpression and Tumorigenicity

A common feature of many oncogenically transformed cells is that theylose the ability of assembling a fibronectin (Fn) matrix. However,exceptions to the rule of neoplastic cells lacking Fn matrix clearlyexist. For example, Fn matrix assembly is dramatically enhanced in hairycell leukemia cells. The specific phenotype (inhibition or stimulationof Fn matrix assembly) is probably determined by the origin of theneoplastic cells and the initial target of the oncogenic transformation.Because Fn matrix has a major impact on cell adhesion, migration, cellgrowth and cell differentiation, an understanding of the molecularmechanism by which cells control Fn matrix assembly may provideimportant information on tumorigenicity and may lead to new ways ofcontrolling tumor growth.

Binding of Fn by specific integrins is critical in initiating Fn matrixassembly. Fn fragments containing the RGD-containing integrin bindingsite or antibodies recognizing the integrin binding site inhibit Fnmatrix assembly. In addition, antibodies to α₅β₁ integrin reduce thedeposition of Fn into extracellular matrix by fibroblasts. In additionto α₅β₁ integrin, members of the β₃ integrins (α.sub.IIb β₃ and α.sub.vβ₃) also initiate Fn matrix assembly, although some of the other Fnbinding integrins such as α₄β₁ or α.sub.v β₁ do not. The ability ofcells to use multiple integrins to support Fn matrix assembly providesthe cells with a versatile mechanism for control of Fn matrix assembly.It may also explain why certain cells, such as fibroblastic cellsderived from α₅ integrin null mutant embryos, assemble a Fn matrix inthe absence of α₅β₁. The primary role of α₅β₁ in Fn matrix assemblyappears to involve initiating the assembly, as Fn mutants lacking theα₅β₁ integrin binding site could not be assembled into Fn matrix unlessin the presence of native Fn.

Activation of specific Fn binding integrins, either by mutations at theintegrin cytoplasmic domains or using activating antibodies,dramatically stimulates Fn matrix assembly. The ability of a cell toassemble a Fn matrix is not only controlled by the types of integrins itexpresses but also regulated by the Fn binding activity of theintegrins. The extracellular ligand binding affinity of integrins can becontrolled from within the cells (inside-out signaling).

Integrin-linked kinase (ILK) may be involved in regulating Fn matrixassembly. ILK binds to the cytoplasmic domains of both β₁ and β₃integrins, and phosphorylates the β₁ cytoplasmic domain in vitro.Overexpression of ILK in epithelial cells dramatically stimulatedintegrin-mediated Fn matrix assembly, down-regulated E-cadherin, andinduced tumor formation in vivo. The data identify ILK as an importantregulator of pericellular Fn matrix assembly, and suggest a criticalrole of this integrin-linked kinase in cell-cell interactions andtumorigenesis.

Reagents

All organic chemicals were of analytic grade and were obtained fromSigma Chemical Co. (St. Louis, Mo.) or Fisher Scientific Co.(Pittsburgh, Pa.) unless otherwise specified. Media for cell culturewere from Gibco Laboratories (Grand Island, N.Y.). Fetal bovine serumwas from HyClone Laboratories, Inc. (Logan, Utah). Polyclonal rabbitanti-β₅ integrin cytoplasmic domain antibody AB47 was generated using asynthetic peptide representing the membrane distal region of the β₅integrin cytoplasmic domain ((SEQ ID NO:11) LPYGTAMEKAQLKPPATSDA).Polyclonal rabbit anti-Fn antibody MC54 was raised against purifiedplasma Fn and purified with a protein A-Sepharose affinity column (Wu etal. (1993) J. Biol. Chem. 268:21883-21888). Polyclonal rabbit anti-29kDa fragment of Fn antibody was raised against the aminoterminal 29 kDafragment of Fn and was further purified using Sepharose beads coupledwith the 29 kDa fragment of Fn (Limper et al. (1991) J Biol. Chem.266:9697-9702). Anti-ILK polyclonal antibody 91-4 was prepared inrabbits as described previously (Hannigan et al. (1996) Nature379:91-96). Monoclonal hamster anti-rat α₅ integrin antibody (HMα5-1)and mouse anti-rat β₃ integrin antibody (F11) were from PharMingen (SanDiego, Calif.). Monoclonal mouse anti-vinculin antibody (hVIN-1) andpurified rabbit IgG were purchased from Sigma (St. Louis, Mo.). The Fnfragments (110 kDa RGD containing integrin binding fragment, the 20 kDaand 70 kDa amino terminal fragments, and the 60 kDa gelatin binding wereprepared as previously described (Quade and McDonald (1988) J. Biol.Chem. 263:19602-19609). cDNA Vectors, Transfection and Cell Culture. Ratintestinal epithelial cells (IEC-18) were maintained in α-MEM medium(Gibco Laboratories, Grand Island, N.Y.) supplemented with 5% FBS(Atlanta Biologicals, Norcross, Ga.), 3.6 mg/ml glucose, 10 μg/mlinsulin and 2 mM glutamine. The pRC/IMV and metallothionein promoter(MT) driven expression vectors containing sense and anti-sense fulllength ILK cDNA sequences were generated as described above. Theexpression vectors were transfected into IEC-18 cells using calciumphosphate and the transfected cells were selected with G418 asdescribed. The expression of human ILK in IEC-18 cells transfected withthe MT-ILK expression vectors (MT-ILK) was induced by growing the cellsin α-MEM medium containing 125 μM ZnSO₄ and 2.5 μM CdCl₂ for 24 to 48hours. The kinase-inactive ILK mutant (GH31R) was generated by a singlepoint mutation (E.fwdarw.K) at amino acid residue 359 within the kinasesubdomain VIII using the Promega Altered Site II in vitro MutagenesisSystem. The mutated DNA was cloned into a pGEX expression system(Pharmacia), and expressed as a GST fusion protein. Kinase assays werecarried out using the recombinant protein as described above and theresults showed that the E³59 .fwdarw.K point mutation completelyinactivated the kinase activity. The cDNA encoding the kinase-inactivemutant was cloned into a pcDNA3 expression vector (Invitrogen),transfected into IEC-18 cells and stable transfectants were selected.

Determination of ILK, E-cadherin and β₁ integrin levels. The cellularlevels of ILK and E-cadherin were determined by immunoblot using anaffinity-purified polyclonal rabbit anti-ILK antibody 91-4, and ananti-E-cadherin antibody (Upstate Biotechnologies, Inc.). The cellsurface expression of α₅β₁ integrins was estimated byimmunoprecipitation of surface biotinylated cell lysates with apolyclonal rabbit anti-α₅β₁ antibody.

Immunofluorescent Staining. Fn matrix assembly was analyzed byimmunofluorescent staining of cell monolayers (Wu et al. (1995) Cell83:715-724). Cells were suspended in the α-MEM medium containing 5% FBSand other additives as specified in each experiment. Cells were platedin 12-well HTC.sup.R slides (Cel-Line, Inc., Newfield, N.J.; 50 μl/well)at a final density of 2×10⁵ cells/ml and cultured in a 37° C. incubatorunder a 5% CO₂-95% air atmosphere. Cells were fixed with 3.7%paraformaldehyde, and staining with the polyclonal rabbit anti-Fnantibody. MC54 (20 μg/ml) and Cy3-conjugated goat anti-rabbit IgGantibodies (Jackson ImmunoResearch Lab, Inc, West Grove, Pa.; 2.5μg/ml). Stained cell monolayers were observed using a Nikon FXAepifluorescence microscope and representative fields were photographedusing Kodak T-Max 400 or Ektachrome 1600 direct positive slide film. Toobtain representative images, exposure times for different experimentalconditions were fixed, using the positive, e.g., matrix forming cells,as the index exposure length.

In double staining experiments, 3.7% paraformaldehyde fixed cells werepermeablized with 0.1% Triton X-100 in TBS containing 1 mg/ml BSA. Thecells were then incubated with primary antibodies from different speciesas specified in each experiment. After rinsing, the bound primaryantibodies were detected with species -specific Cy3- and FITC-conjugatedsecondary antibodies. Stained cell monolayers were observed using aNikon FXA epifluorescence microscope equipped with Cy3 and FITC filters.

For inhibition studies, ILK13-A4a cells that overexpress ILK were platedin 12-well HTC.sup.R slides in the α-MEM medium containing 5% FBS andother additives as specified (2 μM anti-29 kDa Fn fragment antibody, 2μM rabbit control IgG, or 4.2 μM of one of the following Fn fragments:110 kDa RGD containing integrin binding fragment of Fn, 70 kDaaminoterminal fragment of Fn or 60 kDa gelatin binding fragment of Fn).The cells were cultured for four hours, and then fixed and stained withthe polyclonal rabbit anti-Fn antibody and the Cy3-conjugated goatanti-rabbit IgG antibodies as described above.

Isolation and Biochemical Characterization of Extracellular Matrix Fn.To isolate and biochemically characterize extracellular matrix Fn, thecells were cultured in 100 mm tissue culture plates (Corning, Inc.,Corning, N.Y.) in α-MEM medium supplemented with 5% FBS, 2 mML-glutamine, 3.6 mg/ml glucose, 10 μg/ml insulin and other additives asspecified in each experiment for two days. Then the cell monolayers werewashed three times with PBS containing 1 mM AEBSF and harvested with acell scraper. The extracellular matrix fraction was isolated bysequential extraction of the cells with (1) 3% Triton X-100: in PBScontaining 1 mM AEBSF; (2) 100 μg/ml DNase I in 50 mM Tris, pH 7.4, 10mM MnCl₂, 1 M NaC, 1 mM AEBSF and (3) 2% deoxycholate in Tris, pH 8.8, 1mM AEBSF (Wu et al., supra.) Fn in the deoxycholate insolubleextracellular matrix fraction was analyzed by immunoblot with polyclonalrabbit anti-Fn antibody MC54 and an ECL detection kit as previouslydescribed (Wu et al. (1995) J. Cell Sci. 108:821-829).

Colony formation in soft agar. ILK13-Ala3 cells that overexpress ILK(3×10⁵ /well), and Ras-37 cells that overexpress H-RasVal12 (2×10³/well) were plated in 35 mm wells, in 0.3% agarose and assayed forcolony growth after three weeks as described above. Fn fragments wereincorporated in the agar at the final concentrations indicated.

Tumor formation in athymic nude mice. IEC-18, ILK14, or ILK13 cells wereresuspended in PBS and inoculated subcutaneously into athymic nude mice(10.sup.7 /mouse). Six mice were inoculated per cell line. In situ tumorformation was assessed after 3 weeks.

Tyrosine Phosphorylation of p125.sup.FAK in ILK cells. ILK13-A1a3 andILK14-A 2C3 cells growing in monolayer culture were harvested using 5 mMEDTA/PBS (Phosphate Buffered Saline, pH 7.6) and the cells were washedtwice in PBS. Cells were resuspended in serum free medium and thentransferred to plain tissue culture plates (Nunc), tissue culture platesprecoated with 10 μg/ml Fn (Gibco/BRL) or maintained in suspension. Forthe suspension control cells were kept in 50 ml rocker tube. After 1hour incubation at 37° C. in 5% CO₂ cell monolayer (for the adherentcontrols) and cell pellet (for the suspension controls) were washedtwice in ice-cold PBS and lysed in NP-40 lysis buffer (1% NP-40; 150 mMNaCI; 50 mM Tris, pH 7.4; 1 mM EDTA, 1 mM PMSF, 0.2 U/ml aprotonin, 2μg/ml leupeptin and 1 mM Sodium Vanadate). FAK was immunoprecipitatedfrom 400-500 μg of total cell extract using 4 μg mouse monoclonalanti-p125.sup.FAK antibody and Protein A-Agarose conjugate (UBI). Immunecomplexes were washed three times in lysis buffer, boiled in SDS-PAGEsample buffer and run on a 7.5% gel. Resolved proteins were transferredto Immobilon-P (Millipore) and membrane blocked in 5% BSA (Sigma) inTBST (0.1% Tween-20 in Tris Buffered Saline, pH 7.4).Tyrosine-phosphorylated FAK was detected using the RC20H recombinantantibody (HRP-conjugate, Transduction) and ECL detection system(Amersham).

Results

Stimulation of Fn matrix assembly by ILK, To determine whether ILK playsa role in regulation of Fn matrix assembly, the ability of cellsexpressing different levels of ILK to assemble a Fn matrix was analyzed.IEC-18 rat intestinal epithelial cells assembled a small amount of Fnmatrix consisting of mostly short fibrils. ILK13-A1a3 cells, which wereisolated from the IEC-18 cells stably transfected with a pRC/CMVexpression vector containing full length ILK coding sequence, express amuch higher level of ILK than the parental IEC-18 cells. The ILKoverexpressing ILK13-A1a3 cells assembled an extensive Fn matrixresembling that formed by fibroblasts, whereas control transfectants(ILK14-A2C3), which express a similar level of ILK as the parentalIEC-18 cells, assembled a small amount of Fn matrix that isindistinguishable from that of the IEC-18 cells fibroblasts. To excludethe possibility that the observed effect depends on a specific clone,ten additional cell lines were analyzed that were independently isolatedfrom the cells transfected with the pRC/CMV-ILK expression vector(ILK13-A4a, A1d11, A4c, A4c3 and A4i) or the control vector (ILK14-A2C6,A2a3, A2g3, A2g8 and A3a1) Fn matrix assembly was dramatically increasedin all six ILK-overexpressing cell lines (Table 3). On the other hand,all six control cell lines assembled a low level of Fn matrix resemblingthat of the- parental IEC-18 cells. In marked contrast to overexpressionof ILK, overexpression of an oncogenic H-Ras mutant in which the twelfthamino acid residue is mutated (H-RasVal12) in the IEC-18 cells abolishedthe assembly of Fn fibrils.

TABLE 3 Fn matrix assembly by cells expressing different levels of ILKExtra- Cell ILK Expression cellular Fn Line level matrix level ILK13(A1a3, A4a, A1d11, A4c High (wild type ILK) High ILK14 (A2C6, A2C3,A2a3, Low (wild type ILK) Low A2g3, A2g8 and A3a1), IEC-18, MT-ILK6 (E2)GH3:1RH High (kinase-inactive Low mutant)

To further confirm a regulatory role of ILK in Fn matrix assembly,IEC-18 cells were transfected with expression vectors containing fulllength ILK cDNA in the forward (sense) or the reverse (anti-sense)orientation that were under the control of metallothionein promoter(MT). The MT-ILK1 (IIB8) cells, which were derived from the IEC-18 cellstransfected with the sense ILK expression vector, expressed more ILKthan the MT-ILK6 (E2) cells that were derived from the IEC-18 cellstransfected with the anti-sense ILK expression vector. The difference inILK expression was maximized when the cells were grown in the presenceof Zn⁺⁺ and Cd⁺⁺ Consistent with a critical role of ILK in Fn matrixassembly, the ILK overexpressing MT-ILK1 (IIB8) cells exhibit a muchhigh Fn matrix assembly than the MT-ILK6 (E2) cells that have a muchlower level of ILK. Thus, overexpression of ILK, either driven by a CMVpromoter or driven by a metallothionein promoter, stimulates Fn matrixassembly.

Involvement of integrin-linked kinase activity in the cellularregulation of Fn matrix assembly. To test whether the kinase activity isinvolved in the stimulation of Fn matrix assembly by ILK, akinase-inactive ILK mutant (GH31R) was overexpressed in the IEC-18cells. Unlike cells overexpressing the wild type ILK, cellsoverexpressing the kinase-inactive ILK mutant did not assemble anincreased amount of Fn into the extracellular matrix (FIG. 1D). Thus,the kinase activity is critical in the cellular signal transductionleading to the up-regulation of Fn matrix assembly.

Biochemical characterization of Fn matrix assembled by cellsoverexpressing ILK. The Fn matrix deposited by fibroblastic cells ischaracterized by insolubility in sodium deoxycholate. To determinewhether Fn matrix induced by overexpression of ILK in the epithelialcells shares this characteristic, the cell layers were extracted with 2%sodium deoxycholate and the insoluble matrix fractions analyzed byimmunoblotting. The cells overexpressing ILK (A1a3, A4a and IIB8)assembled much more Fn into the deoxycholate insoluble matrix than thecells that express relatively low level of ILK (A2C6, A2C3, and E2). Bycontrast, cells overexpressing H-RasVal12 failed to deposit detectableamount of Fn into the detergent insoluble matrix (H-Ras). These resultsare consistent with the immunofluorescent staining data. Taken together,they provide strong evidence supporting an important role of ILK inregulation of Fn matrix assembly.

Participation of the RGD containing integrin-binding domain and theamino terminal domain of Fn in ILK stimulated Fn matrix assembly.Integrin-mediated Fn matrix assembly requires at least two discreteportions of Fn, the RGD containing integrin-binding domain and theaminoterminal domain. To determine whether these domains alsoparticipate in Fn matrix assembly induced by overexpression of ILK, the110 kDa RGD containing fragment, the 70 kDa aminoterminal domain of Fn,and an antibody against the amino terminal domain of Fn (anti-29 kDa)were utilized. Both the antibody and the Fn fragments decreased the Fnfibril formation induced by ILK. The inhibition was specific, as neitherirrelevant rabbit IgG nor a 60 kDa Fn Fragment lacking the aminoterminus inhibited the Fn matrix assembly. Thus, both the RGD containingintegrin-binding domain and the amino terminal domain of Fn are involvedin Fn matrix assembly promoted by overexpression of ILK, suggesting arole of Fn-binding integrins in this process.

Co-distribution of α5β₁ integrin and Fn matrix in cells overexpressingILK. To begin to identify which Fn-binding integrin mediates Fn matrixassembly induced by overexpression of ILK, cells overexpressing ILK werestained with a hamster monoclonal anti-rat α5 integrin antibody and arabbit polyclonal anti-Fn antibody. The double-staining experimentsshowed that α₅ β₁ integrin was co-localized with Fn fibrils in A1a3cells that overexpress ILK. In contrast, staining of the cells with ananti-rat β₃ integrin antibody revealed no distinctive staining. Theseresults suggest that α₅ β₁ integrin, but not β₃ integrins, participatein the Fn matrix assembly induced by overexpression of ILK.

In contrast to cells that overexpress ILK, cells expressing a lowerlevel of ILK (A2C6) have fewer clusters of α₅β₁ integrin that could bedetected by immunofluorescent staining, although these cells express thesame level of cell surface α₅β₁ integrin as the cells overexpressingILK. Moreover, in marked contrast to A1a3 cells that overexpress ILK,many of the structures containing α₅β₁ integrin in the A2C6 cells lackeddetectable Fn, indicating that overexpression of ILK enhances thebinding of Fn to α₅β₁ integrin.

Effect of ILK overexpression on the formation of focal adhesion andmatrix contacts. Cell adhesion to extracellular substrates is mediatedby transmembrane complexes termed focal adhesions which containintegrin, vinculin and other cytoskeletal proteins. A connection betweenextracellular Fn and the intracellular actin cytoskeleton involving theintegrin β cytoplasmic domain is required for the assembly of Fnfibrils. A2C3 cells that express low levels of ILK formed abundant focaladhesions visualized by staining with an anti-vinculin antibody.However, only a small amount of α₅β₁ integrin and Fn were co-localizedwith the focal adhesions in A2C3 cells.

Overexpression of ILK promoted co-localization of α₅β₁ integrin and Fnwith vinculin. Thus, while cells expressing a relatively low level ofILK are not defective in the assembly of focal adhesion, a higher levelof ILK promotes the assembly of complexes containing vinculin, α₅β₁integrin and Fn matrix. Overexpression of ILK down-regulates E-cadherin.E-cadherin is an important epithelial cell adhesion molecule mediatingcell-cell interactions. Because overexpressing ILK in epithelial cellsdisrupted the characteristic “cobble-stone” epithelial morphology of theepithelial cells, the effect of ILK expression on the cellular level ofE-cadherin was studied. The level of E-cadherin in cells expressingdifferent amount of ILK was determined by immunoblot using ananti-E-cadherin antibody. The parental IEC-18 epithelial cells expressedabundant E-cadherin. Overexpression of H-RasVal12 in IEC-18 cellsreduced the level of E-cadherin. Strikingly, E-cadherin was completelyeliminated in ILK13-A1a3 and A4a cells that overexpress ILK, whereas itwas present at a normal level in ILK14-A2C3 and A2C6 cells that expressa similar level of ILK to the parental IEC-18 cells (FIG. 8A). Theseresults indicate an inverse correlation between the level of ILK andthat of E-cadherin.

In contrast to E-cadherin level, overexpression of ILK did not alter theability of the cells to phosphorylate focal adhesion kinase(pp125.sup.FAK) in response to cell adhesion to Fn, indicating thattyrosine phosphorylation of pp125.sup.FAK does not transduce the signalsleading to the alterations observed upon ILK overexpression, and inparticular tyrosine phosphorylation of pp125.sup.FAK does not play aregulatory role in ILK induced Fn matrix assembly.

Induction of in vivo tumorigenesis by overexpression of ILK. To assess apotential role of ILK in tumorigenesis, cells expressing varying levelsof ILK were injected into athymic nude mice subcutaneously. Tumors arosewithin three weeks in 50% to 100% of the mice injected with the ILK13cells (10.sup.7 cells/mouse) that overexpress ILK, whereas no tumorswere detected in the mice that were injected with the same number of theIEC-18 or ILK14 cells expressing lower levels of ILK (Table 4). Thus,overexpression of ILK in these epithelial cells promotes tumor formationin vivo.

Tumorigenicity of ILK overexpressing IEC-18 Cells Number of Mice withCell Line Tumors at 3 weeks IEC-18 0/6 ILK14-A2C3 0/6 ILK13-A1a3 6/6ILK13-A4a 3/6 Athymic nude mice were inoculated subcutaneously with thecells expressing high (ILK13-Ala3 and A4a) or low (IEC-18 andILK14-A2C3) levels of ILK (10⁷ cells/mouse in PBS). The mice weremonitored for tumor formation at the site of inoculation after threeweeks.

Inhibition of ILK induced cell growth in soft agar by amino terminalfragments of Fn that inhibit matrix assembly. One of the hallmarks oftumor forming cells is that their growth is less dependent on anchorageas measured by their ability to grow in soft agar culture. Similar tocells overexpressing H-Ras, cells overexpressing ILK were able to growin soft agar. However, in marked contrast to the H-Ras overexpressingcells, ILK overexpressing cells assembled an abundant Fn matrix (Table3). It was therefore tested whether the ability of the ILKoverexpressing cells to grow in soft agar culture is related to theelevated level of Fn matrix assembly. The cells overexpressing ILK andthe cells overexpressing H-Ras, respectively, were cultured in soft agareither in the presence or absence of the 70 kDa Fn amino terminalfragment, which inhibits the ILK induced Fn matrix assembly. The 70 kDaFn fragment significantly inhibited the ILK induced “anchorageindependent” growth in soft agar. Similar inhibition was observed withthe 29 kDa fragment of Fn. In contrast, the H-Ras induced anchorageindependent growth in soft agar was not inhibited by the 70 kDa Fnfragmen. Moreover, the ILK induced cell growth in soft agar was notinhibited by the 60 kDa Fn Fragment which does not inhibit the Fn matrixassembly induced by ILK. These results suggest that the cell growth insoft agar induced by ILK, but not that induced by H-Ras, is at leastpartially mediated by a Fn matrix.

Discussion

The overexpression of ILK results in a loss of E-cadherin proteinexpression, offering a possible explanation for the loss of cell-cellcontact in these cells. Indeed, losses of cell-cell adhesion have beenimplicated in tumorigenicity in vivo. ILK overexpressing cells aretumorigenic in nude mice in contrast to the parental IEC-18 intestinalepithelial cells and the control transfected clones. Thus, ILK can beconsidered to be a proto-oncogene. Another important finding is theapparent involvement of ILK in Fn matrix assembly. Overexpression of ILKin IEC-18 cells stimulated Fn matrix assembly. This is a property oftransfected cell clones constitutively overexpressing ILK, and also oftransfected clones in which ILK expression is induced using ametallothionein inducible promoter. Furthermore, Fn matrix assembly isimpaired when an anti-sense ILK cDNA is induced resulting in decreasedILK expression.

The ILK-stimulated Fn matrix assembly was inhibited by theamino-terminal domain of Fn, as well as the RGD-containing integrinbinding domain of Fn, suggesting that RGD-binding integrins mediate ILKfunctions in Fn matrix assembly. Due to the unavailability ofanti-integrin function blocking antibodies against rat integrins, it hasnot been possible to identify directly the specific integrin(s) involvedin the enhanced Fn binding and matrix assembly. However, usingimmunofluorescence analysis, the α₅β₁ integrin, but not α.sub.v β₃, wasco-localized with Fn fibrils in the ILK overexpressing cells,implicating α₅β₁ in the matrix assembly process. Furthermore, ILKoverexpression promoted the co-localization of Fn with α₅β₁ andvinculin, whereas in the parental IEC-18 cells and control transfectedcells vinculin containing focal adhesion plaques were not co-localizedwith Fn.

The kinase activity of ILK is clearly important in the stimulation of Fnmatrix assembly, as overexpression of a kinase-inactive ILK mutantfailed to enhance Fn matrix assembly. However, because ILK has potentialbinding sites for integrins and probably other intracellular signalingmolecules, and because Fn matrix assembly can be regulated by postligand occupancy events, it is possible that other activities of ILK mayalso play important roles in the stimulation of Fn matrix assembly.

Although ILK overexpressing IEC-18 cells express same levels ofintegrins as the parental cells, the ILK overexpressing cells gain theability to grow in an anchorage independent manner in soft agar, and aretumorigenic in nude mice, and they organize a prolific Fn matrix. Thesame IEC-18 cells transfected with an activated form of H-ras, do notassemble a Fn matrix, but nevertheless are highly tumorigenic in nudemice. This represents a novel pathway of oncogenic transformation whichis distinctive from H-Ras induced transformation and involves ILK andenhanced Fn matrix assembly. In fact, the ability to form a Fn matrix isimportant for the anchorage independent growth of transforming growthfactor β (TGF β) treated fibroblasts. Fn matrix assembly also seems tobe important for anchorage-independent growth in soft agar of the ILKoverexpressing cells since inhibition of matrix assembly by the 29 kDaand 70 kDa amino terminal fragments of Fn, results in an inhibition incolony formation in soft agar.

The expression of activated p21.sup.ras results in the disregulation ofmultiple signaling pathways and typically renders cellsserum-independent, as well as anchorage independent for cell growth. Onthe other hand, the overexpression of ILK does not result inserum-independent cell growth, but induces anchorage-independent cellgrowth. These results indicate that ILK normally regulatesadhesion-dependent signaling pathways and that the disregulation of ILK(e.g. by overexpression) induces anchorage-independent cell growthspecifically. It is likely that ILK mediated signaling may be involvedin the regulation of integrin inside-out signaling, as activatedintegrins are required for Fn matrix assembly.

The ability to assemble an extensive Fn fibrillar matrix is a propertyof mesenchymal cells and it is intriguing that the stimulation of thisactivity by ILK overexpression in the epithelial cells is accompanied bya dramatic downregulation of cellular E-cadherin expression. Numerousprevious studies have established that cellular E-cadherin level oractivity is downregulated during epithelial-mesenchymal transition.Moreover, in a recent study, Zuk and Hay demonstrated that inhibition ofα₅β₁ integrin, which is a substrate of ILK, significantly inhibitedepithelial-mesenchymal transition of lens epithelium. It is now alsowidely accepted that many invasive carcinomas exhibit a loss ofE-cadherin expression, and E-cadherin gene has been found to be atumor/invasion-suppressor gene in human lobular breast cancer. The tumorsuppressor gene fat in Drosophila is also homologous to cadherins. ILKmay therefore be involved in coordinating cell-matrix adhesion andcell-cell adhesion in epithelial-mesenchymal transition, andoverexpression of ILK may drive epithelial cells towards a mesenchymalphenotype and oncogenic transformation.

The ILK stimulated Fn matrix assembly may allow enhanced interaction ofFn with α₅β₁. This integrin has recently been shown to be specific insupporting survival of cells on Fn, although no direct correlation wasfound between Fn matrix assembly and α₅β₁ mediated cell survival. Thislatter conclusion was derived from the use of wild type α₅β₁ and α₅cytoplasmic deleted (α₅.DELTA.Cβ₁) mutants. It is likely that for cellsurvival, both receptor interaction with Fn, as well as properintracellular interactions are required. ILK overexpression in IEC-18cells induces cell survival in suspension cultures largely due to theup-regulation of expression of cyclin D₁ and cyclin A proteins.

EXAMPLE 12 Expression of ILK in Human Colon Carcinoma Cells

Tumor (T) or adjacent normal (N) tissue from patients biopsied for coloncarcinoma were analyzed for the expression of ILK or LEF-1 by Westernblot analysis. ILK activity was further determined by an in vitro kinaseassay, as described in previous examples.

These data demonstrate the strong expression of ILK in colon carcinomas,indicating an association with transformation. In accordance with thedata presented in the previous example, LEF-1 expression is closely tiedto ILK expression.

EXAMPLE 13 Phosphoinositide-3-OH Kinase-depehdent Regulation of GSK-3and PKB/AKT by ILK

The amino acid sequence of ILK contains a sequence motif found inpleckstrin homology (PH) domains (Klarulund et al. (1997) Science275:1927-1930). This motif has been shown to be involved in the bindingof phosphatidylinositol phosphates (Lemmon et al. (1996) Cell85:621-624). Amino acids critical to the binding of such lipids to thePH domain are completely conserved in ILK. The phosphatidylinosital3,4,5, triphosphate binding sites are the lysines at positions 162 and209 (SEQ ID NO:2). The PH motifs are comprised of residues 158-165 and208-212 (SEQ ID NO:2). There is a high degree of sequence identitywithin this motif between ILK and other PH-domain containing proteinssuch as cytohesin-1 (a β2 integrin cytoplasmic domain interactingprotein) and GRP-1. It was determined that ILK activity is influenced bythe presence of phosphatidylinosital3,4,5, triphosphate, and interactswith other kinase proteins in this pathway.

Materials and Methods

Stable-Transfected Cells. IEC-18 rat epithelial transformed cells aregrown in Alpha-ME Media with 5% Fetal Calf Serum (GIBCO-BRL), insulin,glucose and L-glutamine. All cells are grown in the absence ofantibiotics and anti-fungal agents. They are harvested and lysed at 80%confluency, with the Lysis Buffer used in the following Kinase Assays.The lysates are quantified with the Bradford Assay.

Transient Transfection. On the day before transfection, the 293 HumanEmbryonic Kidney cells are split such that there will be approximately 1to 1.2 million cells (68% confluent) in a 100 mm (Falcon) dish at thetime of transfection. The cells are fed with DME Media and 10% DonorCalf Serum (GI:BCO-BRL). The cells are grown in the absence ofantibiotics and anti-fungal agents. The use of poly-L-lysine isoptional.

Precipitate plasmids using the calcium/phosphate method with 40 μg ofDNA per dish (15 to 20 μg of plasmids containing ILK construct; 7 to 10μg of plasmids containing GSK-3B construct; use empty vectors whenappropriate), and a 2×HEPES-buffered saline (HeBS) solution of ph 7.05.Allow precipitates to transfect overnight in 3% carbon dioxideenvironment, in 7 ml of DME Media and 5% donor calf serum. The nextmorning, remove the precipitate and medium mixture. Then continue topropagate the cells with 10 ml of DME media and 10% donor calf serumuntil the time of harvest. If the cell become too confluent, they can besplit. Harvest the cell lysates 48 to 60 hours after transfection.

GSK-3B Kinase Assay. Lyse the cells directly from the dish and collectthe cytoplasmic lysate [Lysis Buffer: 150 mM NaCI, 1% NP-40, 0.5% DOC,50 mM ph 7.5 Hepes, 1 μg/ml Leupeptin, 1 μg/ml Aprotinin, 1 mm PMSF and0.1 mM Sodium orthovanadate]. Incubate overnight, 300 μg of pre-clearedcell protein with 1 μl of GSK-3B antibody (Alphonse antibody from JamesWoodgett) in a 500 μl volume. Capture the immunocomplex by incubating 25to 30 μl of protein A-sepharose beads with the lysate for 2 hours at 4degrees centigrade. Collect the beads and wash with cold Lysis Bufferand Kinase Last Wash Buffer, [10 mM Magnesium Chloride, 10 mM ManganeseChloride, 50 mM ph 7.0 Hepes, 0.1 mM Sodium Ortho-Vanadate and 1 mMDTT]. Remove all traces of the supernatant and add 25 μl of KinaseReaction Buffer [50 mM ph 7.0 Hepes, 10 mm Manganese Chloride, 10 mMMagnesium Chloride, 2 mM Sodium Fluoride, 1 mM. Sodium orthovanadate, 1μl of Glycogen Syntase-1 peptide (from James Woodgett)/reaction and 5μMCi/reaction of ATP(gamma 32 phosphate)] to the beads. Incubate themixture for 25 minutes at 30° C. and stop the reaction with the additionof 30 μl of 2× reducing sample buffer. Incubate the mixture at 40° for10 minutes. Do not boil the samples. Run the samples on a Tricine Gel(Schlaeggen and von Jaggow 1987 Anal Biochem 166:368-79) with 15 teeth,1.5 mm Hoefer comb and apparatus overnight at a constant voltage of 110Volts. Visualize the wet gel with a phosphorimager or viaautoradiography.

ILK Kinase Assay. This technique is similar to the GSK-3B Kinase assay.The only differences are the following. For the formation of theimmunocomplex, 1.5-2 μg of antibody is required per sample (200 to 300μg of protein in a 500 to 600 μl volume). The composition of the KinaseReaction Buffer contains 5 μg of myelin basic protein per reactioninstead of the GSK-1 peptide. The reaction is stopped by the addition of30 μl of 2× non-reducing sample buffer, followed by 3 min boiling of thesamples. The samples are separated on a 15% SDS-polyacrylamide gel. Thefixed and dried gel is visualized via autoradiography orphosphorimagery.

Kinase Activation Assessment of Transient Transfected 293 HEK Cells. 48hours after transfection with the various constructs, 293 HEK cells areserum starved, because the cells must be quiescent prior to beingactivated by growth factors. The cultures are washed 3× with serum-freeDMEM and incubated for 12 hrs in serum-free DMEM.

For activating the cell, the serum-free media is removed and thecultures are incubated with DMEM (4 ml per 100 mm dish) supplementedwith the appropriate concentration of growth factors (100 nM Insulin or5 nM IGF-1), and in the presence or absence of a P13 Kinase inhibitor(50 μM LY294002). The activation times vary.

The activation is stopped by washing the cultures 3× with cold PBS,followed by lysing the cells on the dishes with NP-40-DOC lysis buffer.Allow the lysis buffer to work for 30 minutes on ice, before harvesting.Spin the whole cell lysates at 15000 rpms for 15 min and collect thesupernatant. Quantify the supernatant (cytoplasmic lysate) with theBradford Assay. The lysates are now ready to be used forimmunoprecipitation or mixed with 4× sample buffer for Western BlotAnalysis.

Assessment of ILK activation by insulin on IEC-18 cells. IEC 18 cellsare rat colon epithelial cells that are cultured routinely in α-MEMmedium supplemented with insulin (10 mg/liter), glucose 3.6 g/liter, and5% FCS. When IEC 18 cells are grown to 80% confluence, they are serumstarved for 18 hours prior to activation by insulin. Before addition ofinsulin, media are removed and 4 ml of α-MEM+insulin 6 μM is added tothe 100 mm dishes. P13 kinase inhibitors such as LY294002 at 50 μM orwortmannin at 200 nM are added optionally to block P13 kinase dependentILK activation. At the designated times, dishes are washed 3× with icecold PBS and cells are lysed in 500 μl lysis buffer: 150 mM NaCI, 1%NP-40, 0.5% sodium deoxycholate, 50 mM Hepes pH 7.5, 10 μg/ml leupeptin,1 mM PMSF, 2.5 μl aprotinin/mi lysis buffer, NaF 5 mM, Sodium vanadate 1mM. After assessment of protein concentration by Bradford assay, 500 μlsamples containing 200 μg of proteins are incubated for 2 hrs at 4° C.with 20 μl of Protein A-Sepharose to preadsorb the non specific kinases.The lysate is then incubated overnight with 2 μg of rabbit anti ILKantiserum at 4° C. under rotation.

The immunocomplexes are then captured by incubating the lysate with 15μl of Protein A Sepharose for 2 hrs at 4° C. The beads are washed 2×with lysis buffer. The beads are washed 2× with last wash buffer: 10 mMMgCl₂, 10 mM MnCl₂, 50 mM Hepes pH 7.0, 0.1 mM sodium orthovanadate, 2mM NaF, 1 mM DTT. After aspirating completely the buffer, the beads arethen mixed with 25 μl of kinase reaction mixture: 22.5 μl of kinasebuffer (10 mM MgCl₂, 10 mM MnCl₂, 50 mM Hepes pH 7.0, 1 mM sodiumorthovanadate, 2 mM NaF); 2 μl of myelin basic protein at 2 mg/ml (UBI,#13-104), 5 μCi of ³2 P γ-ATP. The kinase reaction is allowed to proceedfor 25 min at 30° C. The reaction is stopped by addition of 30 μl of 2×sample buffer and boiling for 3 min. The samples an then electrophoresedon a 12% SDS-PAGE gel. Phosphorylation level of MBP is assessed byphosphorimager analysis or exposure to an X ray film.

Assessment of ILK kinase activity in 3T3 cells stably transfected withactive or inactive P13 kinase. The cDNAs coding for the HA-tagged P110subunit of the P13 kinase in pcDNA3 were used. 3T3 cells were grown inDMEM with 10% donor calf serum in exponential conditions. The 3T3 cellswere harvested by trypsinization and washed once with HeBs buffer: 20 mMHepes pH 7.05, 137 mM NaCI, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM glucose.10.sup.7 cells were then resuspended in 0.8 ml of ice-cold HeBscontaining 20 μg of uncut DNA. Electroporation was performed with aBio-Rad gene pulser set to 280 V, 960 μF. After electroporation, cellswere allowed to sit on ice for 10 min before being diluted into 24 mlDMEM, 10% DCS and plated on a 150 mm dish. After 2 day recovery,selection was initiated by the addition of G418 at the finalconcentration of 0.8 mg/ml to the culture medium. After 2 weeks, theclones appeared and the transfectants were cloned by serial dilution andculture in 96 well microwell plates. Clones expressing the HA-taggedp110 subunit were expanded and used for ILK kinase assay in serumstarved cells treated with or without with LY 94002.

Transfection of 293 cells protocol. 293 cells have to be exponentiallygrown for optimal transfection. Typically they are passaged every 3 daysby splitting them 1/10. Medium is DMEM medium supplemented with 10%donor calf serum. CaCl₂ M solution: to 14.7 g of CaCl₂2H₂O, add 50 ml ofwater to 50 ml. Filter sterilize through a 0.45 μm nitrocellulosefilter. Store aliquots at −20° C. 2×HBS solution: to 16.4 g NaCI, add11.9 g Hepes and 0.21 g Na₂ HPO₄ and dissolve in 800 Ml H2O. Adjust thepH to 7.12 and add water to 1000 ml. Filter sterilize through a 0.22 μMfilter and store at −20° C. Plasmid solution: 15 μg of ethanolprecipitated plasmids are used per transfection. They are resuspended insterile H₂ O and mixed with 62 μl CaCl₂2M solution. H₂ O is added to 500μl final.

Plate 1×10⁶ 293 cells per 100 mm dish in 10 ml medium 24 h prior totransfection. Mix 50 μl of plasmid solution to 500 μl of 2×HBS solutiondropwise at the same time as bubbling the combined mixture with aPasteur pipette connected to a pipetman. Vortex the mixture for 1 min.and let it stand for 20 min. Add dropwise the 1 ml mixture to the cellsand grow them in 3% CO₂ atmosphere. After 16 hrs of culture, change themedia and grow the cells in normal 5% CO₂ atmosphere. After 48-60 hrs,the cells are harvested for the assay.

Assessment of AKT Phosphorylation by ILK in 293 Cells.

Kinase assay. After cotransfection of 293 cells with HA-AKT and ILK,wild type or kinase dead, the cells are serum starved for 12 hours andsubmitted for activation by growth factors for designated times. Cellsare then lysed with 500 μl lysis buffer: 50 mM Tris-HCl pH 7.4, 0.5%NP-40, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 10 mM 5-glycerophosphate, 0.25mM sodium vanadate, 1 μM microcystin LR, PMSF 1 mM, aprotinin 2.5 μl/ml,leupeptin 10 μg/ml. Prepare a 1:1 slurry of protein G-anti HA mouse Mabbeads as follows: Wash the proteinG.-sepharose beads with solubilizationbuffer 3×. Add 2 μg of anti HA antibody per assay point. Rotate at 4° C.for 1 hr. Wash with solubilization buffer 3×. Resuspend to 1:1 withsolubilization buffer and add 40 μI to the Iysates. Rotate the lysateswith the beads for 1-2 hrs. Wash beads 3 × with solubilization buffercontaining 500 mM NaCl Wash beads 2× with kinase buffer: 20 mM HEPES pH7.4 25 mM β-glycerophosphate, 1 mM sodium vanadate, 1 mM DTT, 1 mMMgCl₂, 1 μM microcystin LR, PMSF and leupeptin.

Aspirate the beads completely. Add 20 μl kinase buffer containing 60 μMCrosstide (From UBI catalog #12-331). Keep cold until ready for kinaseassay. Add 10 μl ATP solution (200 μM cold ATP and 10 μCi/sample ³2 Pγ-ATP in kinase buffer), vortex gently and place tubes in 30° C. waterbath. At 15 min, spot 20 μl onto p81 chromatography paper, let dry forabout 2 min, and immerse into 1% phosphoric acid. Wash blots 6-10×with1% phosphoric acid and count in scintillation counter.

Western blot analysis of AKT (Ser473) phosphorylation state. After cellactivation and lysis, the lysates are mixed with 4× sample buffer andheated to 95-100° C. for 5 minutes and cooled on ice. 20 μl of samplesare run onto SDS-PAGE gels. Proteins are electotransfered on a PVDFmembrane. Incubate membrane in 100 ml blocking buffer, i.e. TBS (Trisbuffered saline) pH. 7.6 supplemented with 5% milk for 1-3 hrs. Incubatemembrane and rabbit anti p-₄73 S AKT antiserum (New England Bio Labs CatNo #9270) at the 1:1000 dilution in 10 ml primary antibody dilutionbuffer with gentle agitation overnight at 4° C.

Primary antibody dilution buffer: 1×TBS, 0.1% Tween 20 with 5% BSA. Wash3 times for 5 minutes each with 15 m TBST. Incubate membrane with horseradish peroxidase (HRP)-conjugated secondary antibody (1:20,000) withgentle agitation for 1 hr at room temperature. Wash membrane 3 times for5 minutes each with 15 m TBST. Incubate membrane with ECL reagent(Amersham) for 1 min at room temperature. Drain membrane of excessdeveloping solution, wrap in Saran wrap and expose to X-ray film.

Assessment of regulation of ILK kinase by phosphoinositides. Ptdlns(3)P,Ptdins(3,4)P₂ and Ptdlns(3,4,5)P₃ were dried under nitrogen andresuspended at 0.1 mM in Hepes 10 mM, pH 7. 0 with phosphatidylserineandphosphatidylcholine, both at 1 mM. The lipid suspensions were vortexedand further sonicated for 20 min in order to generate unilamellarvesicles. 11 μl of ILK5-GST in kinase buffer were combined to 4 μl oflipids and 25 μl of kinase reaction solution containing 2.5 μl of MBPand 5 μCi of γ³2 P-ATP. The reaction proceeded for 30 or 2 hrs at 30° C.The reaction was stopped by adding an equal volume of 2× sample buffer.The samples were run on a 12% non reducing SDS-PAGE gel.

Results

ILK activity is stimulated in vitro by phosphatidylinositol (3,4,5)trisphosphate (Ptdlns(3,4,5)P3) but not by phosphatidylinositol(3,4)bisphosphate (Ptdlns(3,4)P2), or phosphatidylinositol(3) monophosphate(Ptdlns(3)P).

Since Ptdlns(3,4,5,)P3 is specifically generated upon receptor-mediatedstimulation of PI(3)Kinase activity, it was determined whether ILKactivity is stimulated in a PI(3)K dependent manner. PI(3)K is activatedin response to a very wide range of extracellular stimuli, which includegrowth factors and cytokines, as well as by cell adhesion to ECM. ThePtdlns(3,4,5)P3 product of PI(3)K is a second messenger that acts onpathways that control cell proliferation, cell survival, and metabolicchanges often through the activation of P70 ribosomal S6 Kinase(p70.sup.S6k) and protein kinase B (PKB), also known as AKT. PKB/AKT isa protooncogene and has been shown to be activated in a PI(3)K-dependentmanner in response to growth factors, cytokines and cell-ECMinteractions.

To determine whether ILK is activated in a PI(3)K-dependent manner,quiescent, serum-starved, IEC-18 intestinal epithelial cells weretreated with insulin, which is known to activate PI(3)K. ILK activity israpidly stimulated by insulin and this activation is inhibited by priortreatment of the cells with Wortmannin (200 nM), a specific inhibitor ofPI(3)K. Another inhibitor, Ly294002, also inhibits this activation. ILKactivity is rapidly stimulated upon plating cells on fibronectin. Thisactivation is also PI(3)Kinase-dependent, since it is inhibited byLY294002. These data demonstrate that ILK activity is stimulated bygrowth factors, such as insulin, and also by cell-ECM interactions, in aPl(3)K dependent manner, most probably resulting from the directinteraction of PI(3)K generated Ptdlns(3,4,5)P3 with ILK.

To further demonstrate the role of PI(3)K in ILK activation, NIH3T3cells were stably transfected with either constitutively activated P110subunit of PI(3)K, or a kinase-dead mutant of PI(3)K, and ILK activitywas determined in the transfected clones. ILK activity is several-foldhigher in cells expressing constitutively activeP110 subunit of PI(3)K,compared to control cells, or those expressing kinase-dead PI(3)K.Furthermore, the stimulated ILK activity in these cells is inhibited byprior incubation with Ly294002.

Since. ILK overexpression in epithelial cells results in thetranslocation of β-catenin to the nucleus, it was determined whether theactivity of GSK-3, a kinase that normally phosphorylates β-catenin, isregulated by ILK. GSK-3 activity is inhibited when cells encounter Wnt,a matrix associated protein involved in cell fate determination. Theinactivation of GSK-3 results in the inhibition of phosphorylation ofβ-catenin and its subsequent stabilization and nuclear accumulation. ILKmay also contribute to the nuclear localization of β-catenin byinhibiting GSK-3 activity.

Although GSK-3 is expressed in all IEC-18 cell transfectants, itsactivity is dramatically inhibited in the ILK overexpressing ILK-13cells, but not in IEC-18 cells stably expressing a kinase-dead ILK. Asexpected, ILK activity is about 5-fold higher in ILK-13 cells comparedto the control cells. To determine whether this inhibition of GSKactivity is due to ILK, transient transfection assays were carried outin 293 human embryonal kidney epithelial cells. Co-transfection ofHA-tagged-GSK-3 together with wild type ILK results in profoundinhibition of GSK-3 activity, demonstrating that kinase active ILK caninhibit GSK-3 activity. Co-transfection with kinase-dead ILK did notresult in GSK-3 inhibition, but reproducibly resulted in increased GSK-3activity over basal levels. These results suggest that the kinase-deadILK may be acting in a dominant-negative manner by suppressing thefunction of transfected and endogenous ILK.

Since GSK-3 activity can also be regulated by PKB/AKT in aPI(3)K-dependent manner, and since it has previously been shown byothers that integrin engagement stimulates PI(3)K activity leading tothe activation of PKB/AKT, it was determined whether ILK might beupstream of PKB and may regulate its phosphorylation and activation.Co-transfection in 293 cells of HA-tagged PKB with wild-type ILK resultsin specific phosphorylation of PKB on serine473, with the concomitantactivation of its activity. Furthermore, co-transfection withkinase-dead ILK results in a distinct inhibition of serine473phosphorylation, demonstrating again that this form of ILK may becompeting with endogenous ILK and thus behaving in a dominant-negativemanner in the regulation of phosphorylation and activation of PKB. Theidentification of the protein kinases involved in the PI(3)K-mediatedactivation of PKB has been the subject of intense study, and has beenextensively reviewed recently (Downward (1997) Science 279:673-674).Ptdlns(3,4,5)P3 can bind to the PH domain of PKB resulting in itstargeting to the plasma membrane and exposure of threonine-308. Aconstitutively active kinase, PDK-1, then phosphorylates PKB on.threonine-308. However, this phosphorylation alone is not sufficient tofully activate PKB, which also needs to be phosphorylated on serine-473by an as yet unidentified kinase (PDK-2), in a Ptdlns(3,4,5)P3-dependentmanner. The present data shows that ILK, which is activated byPtdlns(3,4,5)P3, can phosphorylate PKB on serine-473, resulting in itsfull activation, thus demonstrating that ILK is directly upstream of PKBin the transduction of PI(3)K-dependent signals to PKB.

In summary, the activity of ILK can be stimulated by Ptdlns(3,4,5)P3 ina PI(3)K-dependent manner and that it can then phosphorylate PKB onserine-473, resulting in its activation. ILK also inactivates GSK-3activity. This inhibition may be indirect, occurring via PKB/AKT, asthis kinase can phosphorylate GSK-3 on serine-9, but it is possible thatILK can also directly phosphorylate GSK-3 and inactivate it,independently of PKB. It will be interesting to determine whether, likeILK, PKB activation also results in the nuclear translocation ofβ-catenin and activation of Lef-1/β-catenin transcriptional activity, orwhether the pathways bifurcate at this point, resulting in ILKactivating the β-catenin pathway, whereas PKB may target other pathwayssuch as P70S6Kinase and control of protein translation or theinactivation of BAD, a pro-apoptotic BcL-2 family member.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 12 <210> SEQ ID NO 1 <211>LENGTH: 1789 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (157)...(1512) <221> NAME/KEY: Other<222> LOCATION: (0)...(0) <400> SEQUENCE: 1 gaattcatct gtcgactgctaccacgggag ttccccggag aaggatcctg cagcccgagt 60 cccgaggata aagcttggggttcatcctcc ttccctggat cactccacag tcctcaggct 120 tccccaatcc aggggactcggcgccgggac gctgct atg gac gac att ttc act 174 Met Asp Asp Ile Phe Thr 15 cag tgc cgg gag ggc aac gca gtc gcc gtt cgc ctg tgg ctg gac aac 222Gln Cys Arg Glu Gly Asn Ala Val Ala Val Arg Leu Trp Leu Asp Asn 10 15 20acg gag aac gac ctc aac cag ggg gac gat cat ggc ttc tcc ccc ttg 270 ThrGlu Asn Asp Leu Asn Gln Gly Asp Asp His Gly Phe Ser Pro Leu 25 30 35 cactgg gcc tgc cga gag ggc cgc tct gct gtg gtt gag atg ttg atc 318 His TrpAla Cys Arg Glu Gly Arg Ser Ala Val Val Glu Met Leu Ile 40 45 50 atg cggggg gca cgg atc aat gta atg aac cgt ggg gat gac acc ccc 366 Met Arg GlyAla Arg Ile Asn Val Met Asn Arg Gly Asp Asp Thr Pro 55 60 65 70 ctg catctg gca gcc agt cat gga cac cgt gat att gta cag aag cta 414 Leu His LeuAla Ala Ser His Gly His Arg Asp Ile Val Gln Lys Leu 75 80 85 ttg cag tacaag gca gac atc aat gca gtg aat gaa cac ggg aat gtg 462 Leu Gln Tyr LysAla Asp Ile Asn Ala Val Asn Glu His Gly Asn Val 90 95 100 ccc ctg cactat gcc tgt ttt tgg ggc caa gat caa gtg gca gag gac 510 Pro Leu His TyrAla Cys Phe Trp Gly Gln Asp Gln Val Ala Glu Asp 105 110 115 ctg gtg gcaaat ggg gcc ctt gtc agc atc tgt aac aag tat gga gag 558 Leu Val Ala AsnGly Ala Leu Val Ser Ile Cys Asn Lys Tyr Gly Glu 120 125 130 atg cct gtggac aaa gcc aag gca ccc ctg aga gag ctt ctc cga gag 606 Met Pro Val AspLys Ala Lys Ala Pro Leu Arg Glu Leu Leu Arg Glu 135 140 145 150 cgg gcagag aag atg ggc cag aat ctc aac cgt att cca tac aag gac 654 Arg Ala GluLys Met Gly Gln Asn Leu Asn Arg Ile Pro Tyr Lys Asp 155 160 165 aca ttctgg aag ggg acc acc cgc act cgg ccc cga aat gga acc ctg 702 Thr Phe TrpLys Gly Thr Thr Arg Thr Arg Pro Arg Asn Gly Thr Leu 170 175 180 aac aaacac tct ggc att gac ttc aaa cag ctt aac ttc ctg acg aag 750 Asn Lys HisSer Gly Ile Asp Phe Lys Gln Leu Asn Phe Leu Thr Lys 185 190 195 ctc aacgag aat cac tct gga gag cta tgg aag ggc cgc tgg cag ggc 798 Leu Asn GluAsn His Ser Gly Glu Leu Trp Lys Gly Arg Trp Gln Gly 200 205 210 aat gacatt gtc gtg aag gtg ctg aag gtt cga gac tgg agt aca agg 846 Asn Asp IleVal Val Lys Val Leu Lys Val Arg Asp Trp Ser Thr Arg 215 220 225 230 aagagc agg gac ttc aat gaa gag tgt ccc cgg ctc agg att ttc tcg 894 Lys SerArg Asp Phe Asn Glu Glu Cys Pro Arg Leu Arg Ile Phe Ser 235 240 245 catcca aat gtg ctc cca gtg cta ggt gcc tgc cag tct cca cct gct 942 His ProAsn Val Leu Pro Val Leu Gly Ala Cys Gln Ser Pro Pro Ala 250 255 260 cctcat cct act ctc atc aca cac tgg atg ccg tat gga tcc ctc tac 990 Pro HisPro Thr Leu Ile Thr His Trp Met Pro Tyr Gly Ser Leu Tyr 265 270 275 aatgta cta cat gaa ggc acc aat ttc gtc gtg gac cag agc cag gct 1038 Asn ValLeu His Glu Gly Thr Asn Phe Val Val Asp Gln Ser Gln Ala 280 285 290 gtgaag ttt gct ttg gac atg gca agg ggc atg gcc ttc cta cac aca 1086 Val LysPhe Ala Leu Asp Met Ala Arg Gly Met Ala Phe Leu His Thr 295 300 305 310cta gag ccc ctc atc cca cga cat gca ctc aat agc cgt agt gta atg 1134 LeuGlu Pro Leu Ile Pro Arg His Ala Leu Asn Ser Arg Ser Val Met 315 320 325att gat gag gac atg act gcc cga att agc atg gct gat gtc aag ttc 1182 IleAsp Glu Asp Met Thr Ala Arg Ile Ser Met Ala Asp Val Lys Phe 330 335 340tct ttc caa tgt cct ggt cgc atg tat gca cct gcc tgg gta gcc ccc 1230 SerPhe Gln Cys Pro Gly Arg Met Tyr Ala Pro Ala Trp Val Ala Pro 345 350 355gaa gct ctg cag aag aag cct gaa gac aca aac aga cgc tca gca gac 1278 GluAla Leu Gln Lys Lys Pro Glu Asp Thr Asn Arg Arg Ser Ala Asp 360 365 370atg tgg agt ttt gca gtg ctt ctg tgg gaa ctg gtg aca cgg gag gta 1326 MetTrp Ser Phe Ala Val Leu Leu Trp Glu Leu Val Thr Arg Glu Val 375 380 385390 ccc ttt gct gac ctc tcc aat atg gag att gga atg aag gtg gca ttg 1374Pro Phe Ala Asp Leu Ser Asn Met Glu Ile Gly Met Lys Val Ala Leu 395 400405 gaa ggc ctt cgg cct acc atc cca cca ggt att tcc cct cat gtg tgt 1422Glu Gly Leu Arg Pro Thr Ile Pro Pro Gly Ile Ser Pro His Val Cys 410 415420 aag ctc atg aag atc tgc atg aat gaa gac cct gca aag cga ccc aaa 1470Lys Leu Met Lys Ile Cys Met Asn Glu Asp Pro Ala Lys Arg Pro Lys 425 430435 ttt gac atg att gtg cct atc ctt gag aag atg cag gac aag 1512 Phe AspMet Ile Val Pro Ile Leu Glu Lys Met Gln Asp Lys 440 445 450 taggactggaaggtccttgc ctgaactcca gaggtgtcgg gacatggttg ggggaatgca 1572 cctccccaaagcagcaggcc tctggttgcc tcccccgcct ccagtcatgg tactacccca 1632 gcctggggtccatccccttc ccccatccct accactgtgc gcaagagggg cgggctcaga 1692 gctttgtcacttgccacatg gtgtctccca acatgggagg gatcagcccc gcctgtcaca 1752 ataaagtttattatgaaaaa aaaaaaaaaa aaaaaaa 1789 <210> SEQ ID NO 2 <211> LENGTH: 452<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met AspAsp Ile Phe Thr Gln Cys Arg Glu Gly Asn Ala Val Ala Val 1 5 10 15 ArgLeu Trp Leu Asp Asn Thr Glu Asn Asp Leu Asn Gln Gly Asp Asp 20 25 30 HisGly Phe Ser Pro Leu His Trp Ala Cys Arg Glu Gly Arg Ser Ala 35 40 45 ValVal Glu Met Leu Ile Met Arg Gly Ala Arg Ile Asn Val Met Asn 50 55 60 ArgGly Asp Asp Thr Pro Leu His Leu Ala Ala Ser His Gly His Arg 65 70 75 80Asp Ile Val Gln Lys Leu Leu Gln Tyr Lys Ala Asp Ile Asn Ala Val 85 90 95Asn Glu His Gly Asn Val Pro Leu His Tyr Ala Cys Phe Trp Gly Gln 100 105110 Asp Gln Val Ala Glu Asp Leu Val Ala Asn Gly Ala Leu Val Ser Ile 115120 125 Cys Asn Lys Tyr Gly Glu Met Pro Val Asp Lys Ala Lys Ala Pro Leu130 135 140 Arg Glu Leu Leu Arg Glu Arg Ala Glu Lys Met Gly Gln Asn LeuAsn 145 150 155 160 Arg Ile Pro Tyr Lys Asp Thr Phe Trp Lys Gly Thr ThrArg Thr Arg 165 170 175 Pro Arg Asn Gly Thr Leu Asn Lys His Ser Gly IleAsp Phe Lys Gln 180 185 190 Leu Asn Phe Leu Thr Lys Leu Asn Glu Asn HisSer Gly Glu Leu Trp 195 200 205 Lys Gly Arg Trp Gln Gly Asn Asp Ile ValVal Lys Val Leu Lys Val 210 215 220 Arg Asp Trp Ser Thr Arg Lys Ser ArgAsp Phe Asn Glu Glu Cys Pro 225 230 235 240 Arg Leu Arg Ile Phe Ser HisPro Asn Val Leu Pro Val Leu Gly Ala 245 250 255 Cys Gln Ser Pro Pro AlaPro His Pro Thr Leu Ile Thr His Trp Met 260 265 270 Pro Tyr Gly Ser LeuTyr Asn Val Leu His Glu Gly Thr Asn Phe Val 275 280 285 Val Asp Gln SerGln Ala Val Lys Phe Ala Leu Asp Met Ala Arg Gly 290 295 300 Met Ala PheLeu His Thr Leu Glu Pro Leu Ile Pro Arg His Ala Leu 305 310 315 320 AsnSer Arg Ser Val Met Ile Asp Glu Asp Met Thr Ala Arg Ile Ser 325 330 335Met Ala Asp Val Lys Phe Ser Phe Gln Cys Pro Gly Arg Met Tyr Ala 340 345350 Pro Ala Trp Val Ala Pro Glu Ala Leu Gln Lys Lys Pro Glu Asp Thr 355360 365 Asn Arg Arg Ser Ala Asp Met Trp Ser Phe Ala Val Leu Leu Trp Glu370 375 380 Leu Val Thr Arg Glu Val Pro Phe Ala Asp Leu Ser Asn Met GluIle 385 390 395 400 Gly Met Lys Val Ala Leu Glu Gly Leu Arg Pro Thr IlePro Pro Gly 405 410 415 Ile Ser Pro His Val Cys Lys Leu Met Lys Ile CysMet Asn Glu Asp 420 425 430 Pro Ala Lys Arg Pro Lys Phe Asp Met Ile ValPro Ile Leu Glu Lys 435 440 445 Met Gln Asp Lys 450 <210> SEQ ID NO 3<211> LENGTH: 452 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: Other <222> LOCATION: (0)...(0) <400> SEQUENCE:3 Met Asp Asp Ile Phe Thr Gln Cys Arg Glu Gly Asn Ala Val Ala Val 1 5 1015 Arg Leu Trp Leu Asp Asn Thr Glu Asn Asp Leu Asn Gln Gly Asp Asp 20 2530 His Gly Phe Ser Pro Leu His Trp Ala Cys Arg Glu Gly Arg Ser Ala 35 4045 Val Val Glu Met Leu Ile Met Arg Gly Ala Arg Ile Asn Val Met Asn 50 5560 Arg Gly Asp Asp Thr Pro Leu His Leu Ala Ala Ser His Gly His Arg 65 7075 80 Asp Ile Val Gln Lys Leu Leu Gln Tyr Lys Ala Asp Ile Asn Ala Val 8590 95 Asn Glu His Gly Asn Val Pro Leu His Tyr Ala Cys Phe Trp Gly Gln100 105 110 Asp Gln Val Ala Glu Asp Leu Val Ala Asn Gly Ala Leu Val SerIle 115 120 125 Cys Asn Lys Tyr Gly Glu Met Pro Val Asp Lys Ala Lys AlaPro Leu 130 135 140 Arg Glu Leu Leu Arg Glu Arg Ala Glu Lys Met Gly GlnAsn Leu Asn 145 150 155 160 Arg Ile Pro Tyr Lys Asp Thr Phe Trp Lys GlyThr Thr Arg Thr Arg 165 170 175 Pro Arg Asn Gly Thr Leu Asn Lys His SerGly Ile Asp Phe Lys Gln 180 185 190 Leu Asn Phe Leu Thr Lys Leu Asn GluAsn His Ser Gly Glu Leu Trp 195 200 205 Lys Gly Arg Trp Gln Gly Asn AspIle Val Val Lys Val Leu Lys Val 210 215 220 Arg Asp Trp Ser Thr Arg LysSer Arg Asp Phe Asn Glu Glu Cys Pro 225 230 235 240 Arg Leu Arg Ile PheSer His Pro Asn Val Leu Pro Val Leu Gly Ala 245 250 255 Cys Gln Ser ProPro Ala Pro His Pro Thr Leu Ile Thr His Trp Met 260 265 270 Pro Tyr GlySer Leu Tyr Asn Val Leu His Glu Gly Thr Asn Phe Val 275 280 285 Val AspGln Ser Gln Ala Val Lys Phe Ala Leu Asp Met Ala Arg Gly 290 295 300 MetAla Phe Leu His Thr Leu Glu Pro Leu Ile Pro Arg His Ala Leu 305 310 315320 Asn Ser Arg Ser Val Met Ile Asp Glu Asp Met Thr Ala Arg Ile Ser 325330 335 Met Ala Asp Val Lys Phe Ser Phe Gln Cys Pro Gly Arg Met Tyr Ala340 345 350 Pro Ala Trp Val Ala Pro Glu Ala Leu Gln Lys Lys Pro Glu AspThr 355 360 365 Asn Arg Arg Ser Ala Asp Met Trp Ser Phe Ala Val Leu LeuTrp Glu 370 375 380 Leu Val Thr Arg Glu Val Pro Phe Ala Asp Leu Ser AsnMet Glu Ile 385 390 395 400 Gly Met Lys Val Ala Leu Glu Gly Leu Arg ProThr Ile Pro Pro Gly 405 410 415 Ile Ser Pro His Val Cys Lys Leu Met LysIle Cys Met Asn Glu Asp 420 425 430 Pro Ala Lys Arg Pro Lys Phe Asp MetIle Val Pro Ile Leu Glu Lys 435 440 445 Met Gln Asp Lys 450 <210> SEQ IDNO 4 <211> LENGTH: 256 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: Other <222> LOCATION: (1)...(256) <400>SEQUENCE: 4 Ile Pro Arg Glu Ser Leu Arg Leu Glu Val Lys Leu Gly Gln GlyCys 1 5 10 15 Phe Gly Glu Val Trp Met Gly Thr Trp Asn Gly Thr Thr LysVal Ala 20 25 30 Ile Lys Thr Leu Lys Pro Gly Thr Met Met Pro Glu Ala PheLeu Gln 35 40 45 Glu Ala Gln Ile Met Lys Lys Leu Arg His Asp Lys Leu ValPro Leu 50 55 60 Tyr Ala Val Val Ser Glu Glu Pro Ile Tyr Ile Val Thr GluPhe Met 65 70 75 80 Thr Lys Gly Ser Leu Leu Asp Phe Leu Lys Glu Gly GluGly Lys Phe 85 90 95 Leu Lys Leu Pro Gln Leu Val Asp Met Ala Ala Gln IleAla Asp Gly 100 105 110 Met Ala Tyr Ile Glu Arg Met Asn Tyr Ile His ArgAsp Leu Arg Ala 115 120 125 Ala Asn Ile Leu Val Gly Asp Asn Leu Val CysLys Ile Ala Asp Phe 130 135 140 Gly Leu Ala Arg Leu Ile Glu Asp Asn GluTyr Thr Ala Arg Gln Gly 145 150 155 160 Ala Lys Phe Pro Ile Lys Trp ThrAla Pro Glu Ala Ala Leu Tyr Gly 165 170 175 Arg Phe Thr Ile Lys Ser AspVal Trp Ser Phe Gly Ile Leu Leu Thr 180 185 190 Glu Leu Val Thr Lys GlyArg Val Pro Tyr Pro Gly Met Val Asn Arg 195 200 205 Glu Val Leu Glu GlnVal Glu Arg Gly Tyr Arg Met Pro Cys Pro Gln 210 215 220 Gly Cys Pro GluSer Leu His Glu Leu Met Lys Leu Cys Trp Lys Lys 225 230 235 240 Asp ProAsp Glu Arg Pro Thr Phe Glu Tyr Ile Gln Ser Phe Leu Glu 245 250 255<210> SEQ ID NO 5 <211> LENGTH: 263 <212> TYPE: PRT <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: Other <222> LOCATION: (1)...(263)<400> SEQUENCE: 5 Ile Pro Trp Cys Asp Leu Asn Ile Lys Glu Lys Ile GlyAla Gly Ser 1 5 10 15 Phe Gly Thr Val His Arg Ala Glu Trp His Gly SerAsp Val Ala Val 20 25 30 Lys Ile Leu Met Glu Gln Asp Phe His Ala Glu ArgVal Asn Glu Phe 35 40 45 Leu Arg Glu Val Ala Ile Met Lys Arg Leu Arg HisPro Asn Ile Val 50 55 60 Leu Phe Met Gly Ala Val Thr Gln Pro Pro Asn LeuSer Ile Val Thr 65 70 75 80 Glu Tyr Leu Ser Arg Gly Ser Leu Tyr Arg LeuLeu His Lys Ser Gly 85 90 95 Ala Arg Glu Gln Leu Asp Glu Arg Arg Arg LeuSer Met Ala Tyr Asp 100 105 110 Val Ala Lys Gly Met Asn Tyr Leu His AsnArg Asn Pro Pro Ile Val 115 120 125 His Arg Asp Leu Lys Ser Pro Asn LeuLeu Val Asp Lys Lys Tyr Thr 130 135 140 Val Lys Val Cys Asp Phe Gly LeuSer Arg Leu Lys Ala Ser Thr Phe 145 150 155 160 Leu Ser Ser Lys Ser AlaAla Gly Thr Pro Glu Trp Met Ala Pro Glu 165 170 175 Val Leu Arg Asp GluPro Ser Asn Glu Lys Ser Asp Val Tyr Ser Phe 180 185 190 Gly Val Ile LeuTrp Glu Leu Ala Thr Leu Gln Gln Pro Trp Gly Asn 195 200 205 Leu Asn ProAla Gln Val Val Ala Ala Val Gly Phe Lys Cys Lys Arg 210 215 220 Leu GluIle Pro Arg Asn Leu Asn Pro Gln Val Ala Ala Ile Ile Glu 225 230 235 240Gly Cys Trp Thr Asn Glu Pro Trp Lys Arg Pro Ser Phe Ala Thr Ile 245 250255 Met Asp Leu Leu Arg Pro Leu 260 <210> SEQ ID NO 6 <211> LENGTH: 271<212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: Other <222> LOCATION: (1)...(271) <400> SEQUENCE: 6 Ile ProAsp Gly Gln Ile Thr Val Gly Gln Arg Ile Gly Ser Gly Ser 1 5 10 15 PheGly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val Ala Val Lys 20 25 30 MetLeu Asn Val Thr Ala Pro Thr Pro Gln Gln Leu Gln Ala Phe Lys 35 40 45 AsnGlu Val Gly Val Leu Arg Lys Thr Arg His Val Asn Ile Leu Leu 50 55 60 PheMet Gly Tyr Ser Thr Lys Pro Gln Leu Ala Ile Val Thr Gln Trp 65 70 75 80Cys Glu Gly Ser Ser Leu Tyr His His Leu His Ile Ile Glu Thr Lys 85 90 95Phe Glu Met Ile Lys Leu Ile Asp Ile Ala Arg Gln Thr Ala Gln Gly 100 105110 Met Asp Tyr Leu His Ala Lys Ser Ile Ile His Arg Asp Leu Lys Ser 115120 125 Asn Asn Ile Phe Leu His Glu Asp Leu Thr Val Lys Ile Gly Asp Phe130 135 140 Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser His Gln PheGlu 145 150 155 160 Gln Leu Ser Gly Ser Ile Leu Trp Met Ala Pro Glu ValIle Arg Met 165 170 175 Gln Asp Lys Asn Pro Tyr Ser Phe Gln Ser Asp ValTyr Ala Phe Gly 180 185 190 Ile Val Leu Tyr Glu Leu Met Thr Gly Gln LeuPro Tyr Ser Asn Ile 195 200 205 Asn Asn Arg Asp Gln Ile Ile Phe Met ValGly Arg Gly Tyr Leu Ser 210 215 220 Pro Asp Leu Ser Lys Val Arg Ser AsnCys Pro Lys Ala Met Lys Arg 225 230 235 240 Leu Met Ala Glu Cys Leu LysLys Lys Arg Asp Glu Arg Pro Leu Phe 245 250 255 Pro Gln Ile Leu Ala SerIle Glu Leu Leu Ala Arg Ser Leu Pro 260 265 270 <210> SEQ ID NO 7 <211>LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: Other <222> LOCATION: (1)...(31) <400> SEQUENCE: 7ggccgaattc gctggaattg ttcttattgg c 31 <210> SEQ ID NO 8 <211> LENGTH: 31<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: Other <222> LOCATION: (1)...(31) <400> SEQUENCE: 8 ggccggatcctcattttccc tcatacttcg g 31 <210> SEQ ID NO 9 <211> LENGTH: 32 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 9 ccttcagcaccctcacgaca atgtcattgc cc 32 <210> SEQ ID NO 10 <211> LENGTH: 32 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 10 ctgcagagctttgggggcat cccaggcagg tg 32 <210> SEQ ID NO 11 <211> LENGTH: 20 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 11 Leu Pro TyrGly Thr Ala Met Glu Lys Ala Gln Leu Lys Pro Pro Ala 1 5 10 15 Thr SerAsp Ala 20 <210> SEQ ID NO 12 <211> LENGTH: 7080 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: exon <222>LOCATION: (1)...(41) <221> NAME/KEY: exon <222> LOCATION: (401)...(581)<221> NAME/KEY: exon <222> LOCATION: (4260)...(4425) <221> NAME/KEY:exon <222> LOCATION: (4608)...(4703) <221> NAME/KEY: exon <222>LOCATION: (4908)...(5004) <221> NAME/KEY: exon <222> LOCATION:(5098)...(5181) <221> NAME/KEY: exon <222> LOCATION: (5285)...(5370)<221> NAME/KEY: exon <222> LOCATION: (5513)...(5622) <221> NAME/KEY:exon <222> LOCATION: (5726)...(5853) <221> NAME/KEY: exon <222>LOCATION: (5938)...(6059) <221> NAME/KEY: exon <222> LOCATION:(6150)...(6249) <221> NAME/KEY: exon <222> LOCATION: (6362)...(6492)<221> NAME/KEY: exon <222> LOCATION: (6676)...(7080) <400> SEQUENCE: 12acgggagttc cccggagaag gatcctgcag cccgagtccc ggtgagtgcg agaggacccg 60cgccccctgt cacccctcac ttctcctgag tcccaagtaa ggaacggagc caacccttgg 120ggaggacccc ccggtcccat ctcccagagt atacggagcc tgaacctccc cacttccccc 180aactctgatc gcggataggg tctagttgcc tgctctcgga catccgttca gcagacacta 240cctcttcgtc accccctgcc caccctgacc cgcctttacc tcgcgtctag aggacacagc 300caggggtcat cccgcagccc cgaacacctt cacagacccc cactagccgg ggacgcagct 360caggccccct acccccaaca caaacacttc tctcctgtag aggataaagc ttggggttca 420tcctccttcc ctggatcact ccacagtcct caggcttccc caatccaggg gactcggcgc 480cgggacgctg ctatggacga cattttcact cagtgccggg agggcaacgc agtcgccgtt 540cgcctgtggc tggacaacac ggagaacgac ctcaaccagg ggtgagctga aacggttggt 600ggatgagagg aaggctagag atctcctggc tacgtggagt ggagtgctca tgtctggtgg 660tggcggctgc ctttgctagc cagtgtaatg agtgctaagc atagcctgtg gggggcagag 720ggttttcaca gaggacgcag tttgagctga atcttgactg cagaataaga gtttcacagg 780cagaggctac agagagcttt ccagcctcag tagactgcat ggccaaaggc tagggggcaa 840ggcacagagc agagagaaca ggaaattgtc tggagttcca tatggctgga ggatagatga 900tctctgtggg tcggagcatt gggtacaaga ggaaaagaac aggacataag actgtagagg 960tctccaagga tcagccagat catgcaagca tatttaggct ataagaaggt ctttatttta 1020aaagcaaggt tttaaagctg tatagtgaca taatgagatt gtccattttt cagtatcgtc 1080ttggatgctt tgtgaagagc aggtggaaag gaggcaattg cctagttcat cgtagaagta 1140atgatgtctt ggactagaat tagggtgagt gaaaaagaga agtgatcaaa ttccagaagt 1200ctttagggac gaagaattgt ggaacttggt gtggggagtg agtaagggca aagatgagga 1260ccagggttct ggcttgggaa cttggttgtt tttactgaga tggagaaaac tggaggatga 1320ggcattttat tttggacatg ttaaagttaa ggtgacaata cagcaaggtg gaaatgttga 1380gtgtgttgtt ggttatgcag atttgcaatt caggggagga atctgacttg gagacacaga 1440tttgagagct atcagcatat agatagcata tatatatcat atatatagat gatacttgaa 1500gacgtggttg tgaaggagaa cgtctcagga gagtggaagg caagaggtca ggggacagga 1560agcctgcacg acagaaaagg agcaaccgca agttgaaaaa gaaataggaa aatgagtaaa 1620gcttacagaa gagaatgttt taatgaaggg gaggtagtca acggcagtac aggctactga 1680gagatcaaag aacattaata ttttaaagta gccattgaat ttacagttgg gttttttttt 1740tttaagttaa ggggtacatg tgcaggtttg ttggatgggt aaactcaggt catggaggtt 1800tgttgtgcag atcatttcat cacccaggta ttaagccgtg gcacccatta gttatttttc 1860ctgatccctc cctcttccta cccgccccac tctggtaggc cccagtgtct gttgctcccc 1920tcagtgtgca gttaggatgt atttgaccat ctgtgagtgt tgtttcagaa taatgggtga 1980agaaatcaga ctacattggg ttccttaatt taaggcacta tgatgaacac cttctgtgct 2040gggtgctaga gtccagaatc tgacataatc tctgctctcg atacagcaga tttgtggcag 2100attgaaaagt aggccgggcg cggtgctcac gcctgtaatc ccagcacttt gggaggccga 2160ggctggtgga tcacgaggtc aggagttcga gaccagcctg accaacatgg tgaaaccctg 2220tctctactaa aaatacaaaa attagccagg cgtggtgcgt gcgcctgtaa tcccagctac 2280tcggaggctg aggcaggaga actgcttgaa cccgggaagc agaggctgca gtgagccaag 2340atcatgccac tgccctccag cctgggccac agagcaagac tctgtcttaa aaaagaaaaa 2400gaaaacaaaa gttgagaggt gagcaaatag tgaaagtgaa tgtacaccag ttctacaagg 2460ttggctgaga taggaaggca agaagagcag tagtgatggt gactcagagt ggcaaaatca 2520aattctttat tttttaaaat tggtggagtt tagtttttag gtttgacagg tcaggatcac 2580ctgtcatctt agtctagact tcactggttc ccattaggtt ctacactatt tctctttaat 2640actttgcaaa gtcatttgtt ccatttttca tataattctc aaagggtacc ctgataaaga 2700cagtaagcac tgttactgtt ttgttggtga ggaaatagag gcttaggcag ttgcccagtg 2760ggcctagcat ctaggaagac tctagctgag tctaggaaga ctctaggaag atgctgccag 2820aaggaaaagg ggtggaatta atgaaactgg aaggttgtgg tgctggtttg aggagtaaag 2880tatgggggcc aaagttggct atatgctgga tatgaagagg gggttaattc ttgcaggtct 2940tcttgagata gaagtccagg ccctgaggtg gcacgcagcc tgatagtgaa cagaaccctt 3000gtgcccatac tttgcccccc gagtttggcc tgccctagcg ccacaggcag tactggaagc 3060tgataagtca gggcacaaat taaaggtcta ggaggcaaga ccaactgata aacagggggc 3120gtaatgcttc ctggggataa tcccatctca aggaaaagct tttggctatt tctgtgttag 3180cagatgcgag caagttgtgg aggaaaatga ttaacttaaa ggccacaccc cccaacccct 3240tcagctgtac gggcatctgg atggccagac ccctgatcag ctccaggcct tgcctagagt 3300cgcacacagg aagtgaccac aacactaagg catccggagg ctgctggccc agaccacgga 3360ccgccccctg ctccgcccct tgaatctgca gccccgaact ggagggaatt tccttttatg 3420gccccaggct gtcaggcagg cccagctcca aaccaattgt tttgttttgt ttgtatactt 3480tactttataa aatacatatt tcttggaaaa gaaaaaaatc aggtatagtt ttaatcccaa 3540aacagccatt ctcattttct tctgttttct atgcacagga ttgctaagta ggggaatcct 3600gctttgcatg gtggtttgtt atctacctca tcagtagtca aaagcatttc tccttttcaa 3660cataaaccct tccaaaacat catttaaatt ccattccatt tttagatgtt agtttcagta 3720ccagagccaa atagtaagtt tgttccctct tctataagtt gtggcctgcc ttctccaaga 3780cacccttctc ccccactaac atgcactgaa cagctgctgt caatccattc cctcaagaaa 3840cttaaaggtc taacagacaa gacagtccat taacagattt ttacaatcca gtgcaacaag 3900tgctgaagta gggggacata taagatgtgg tggaaaacag aaaggaccaa tagatgttgc 3960agaaggggtg caagagaaac caggggaaga gcactaccac ctaaggaaat gagatgtggg 4020atatggtgaa gataacacat ttttttatag aggttgttga gatatctagg tggttggttt 4080ggtttgaagc tcaggggaag gtctgaaatg gacagctttg ggagttgctg gcatgaatga 4140gaatcaaagt cctagagagg taagccttcc cagagagtat gtaattatca gtttttcagg 4200aatcaaaacc tttgccccat cccacctcca gctcaatgac cattgcccct tcctcaaagg 4260gacgatcatg gcttctcccc cttgcactgg gcctgccgag agggccgctc tgctgtggtt 4320gagatgttga tcatgcgggg ggcacggatc aatgtaatga accgtgggga tgacaccccc 4380ctgcatctgg cagccagtca tggacaccgt gatattgtac agaaggtacg tacaaactcc 4440ttcgtcatcc acatcacata catgccatga gggtcagtca caggcactgt aacatacagt 4500agaaagcatg tgtgctcttc ccccttttcc catgccctga caccagtatc tcatttggaa 4560ctgactgtac tttctgcctc ttctttttgt ctggccatgg ggtccagcta ttgcagtaca 4620aggcagacat caatgcagtg aatgaacacg ggaatgtgcc cctgcactat gcctgttttt 4680ggggccaaga tcaagtggca gaggtgagta ctcagccctt aattcctgag atgggtagga 4740agtaaagtct gagccttggt gggagatttt ggaaccctca acccattctg tcagtactac 4800tgtgtgacac ttacaggatt aagttctaca tttgtgcatt catggttggt tcagtgactg 4860ccagcgaggt agcagtggct ctcatcataa tggccttttc attccaggac ctggtggcaa 4920atggggccct tgtcagcatc tgtaacaagt atggagagat gcctgtggac aaagccaagg 4980cacccctgag agagcttctc cgaggtccat ctccccatcc cctagcttgt gtcctctcgt 5040cccttcccac ctgtcttctc cctctgtacc acagcttagg ttgtttttct tccctagagc 5100gggcagagaa gatgggccag aatctcaacc gtattccata caaggacaca ttctggaagg 5160ggaccacccg cactcggccc cgtgagtcac cactgtggga agaagggttg taaaaggaaa 5220taatcctggc ctcttggggc tgggttaggg tgaagctggg tacctgacct gcccacactc 5280ttaggaaatg gaaccctgaa caaacactct ggcattgact tcaaacagct taacttcctg 5340acgaagctca acgagaatca ctctggagag gtgacccctg cccttcttgc ccttccctca 5400ctaaaccccc ataaattact tgctttgtac ctgttttaag tttttcctcc agttagtggg 5460caaggaagtg gcagcaacat ttcaagcctc ctaaccccta cctgtcctgc agctatggaa 5520gggccgctgg cagggcaatg acattgtcgt gaaggtgctg aaggttcgag actggagtac 5580aaggaagagc agggacttca atgaagagtg tccccggctc aggtagtgca aggcgtaacc 5640tggaagctgc tagttccaag gaaccctgaa tagcactgaa agagatcttt tgtactgggt 5700ctcaaccact ccctccctct tctaggattt tctcgcatcc aaatgtgctc ccagtgctag 5760gtgcctgcca gtctccacct gctcctcatc ctactctcat cacacactgg atgccgtatg 5820gatccctcta caatgtacta catgaaggca ccagtgagta gggatgttga atttccttgg 5880ggaggaaatg gcagagaggg agcctctctg aactatttga cttttgcctc ctctcagatt 5940tcgtcgtgga ccagagccag gctgtgaagt ttgctttgga catggcaagg ggcatggcct 6000tcctacacac actagagccc ctcatcccac gacatgcact caatagccgt agtgtaatgg 6060tgaggccaca agctcactcc tggcccaggc cccaaaagcc ctttgcctat ctatgactta 6120cctccttcta tctgttttct cttcctcaga ttgatgagga catgactgcc cgaattagca 6180tggctgatgt caagttctct ttccaatgtc ctggtcgcat gtatgcacct gcctgggtag 6240cccccgaagg tgagtgaagt catcatgtcg ggaggtaaaa aaggaccacc tcagaagtag 6300tggaaggggg cagagacagg acaggcaagg gggccagaac agacaagccc tatctctcca 6360gctctgcaga agaagcctga agacacaaac agacgctcag cagacatgtg gagttttgca 6420gtgcttctgt gggaactggt gacacgggag gtaccctttg ctgacctctc caatatggag 6480attggaatga aggtgagagc acaacagcat acatttgtgt tgcgggagtg gttggtgatg 6540gtgaaaataa ctgtagtggg ccttggctcc tcacatattt gttcggatat acagtaatcc 6600tgtcccaagg gccagtggct tctctctaca tgacagactc aaattgtgag gctgcttttt 6660ttcttgtatt cgcaggtggc attggaaggc cttcggccta ccatcccacc aggtatttcc 6720cctcatgtgt gtaagctcat gaagatctgc atgaatgaag accctgcaaa gcgacccaaa 6780tttgacatga ttgtgcctat ccttgagaag atgcaggaca agtaggactg gaaggtcctt 6840gcctgaactc cagaggtgtc gggacatggt tgggggaatg cacctcccca aagcagcagg 6900cctctggttg cctcccccgc ctccagtcat ggtactaccc cagcctgggg tccatcccct 6960tcccccatcc ctaccactgt gcgcaagagg ggcgggctca gagctttgtc acttgccaca 7020tggtgtcttc caacatggga gggatcagcc ccgcctgtca caataaagtt tattatgaaa 7080

What is claimed is:
 1. An isolated nucleic acid other than an intactchromosome, wherein said nucleic acid comprises the sequence set forthin SEQ ID NO:12.